Trail receptors, nucleic acids encoding the same, and methods of use thereof

ABSTRACT

In accordance with the present invention, there are provided isolated mammalian TRAIL receptor proteins, antibodies thereto, therapeutic compositions, and nucleic acids encoding such. Bioassays and therapeutic methods employing invention DR5 and TRAIL-R3 proteins are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. provisionalapplication serial No. 60/055,906, filed Aug. 15, 1997, now abandoned.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grantnumber AG 13487 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND INFORMATION

[0003] Apoptosis is a normal physiological process of cell death thatplays a critical role in the regulation of tissue homeostasis byensuring that the rate of new cell accumulation produced by celldivision is offset by a commensurate rate of cell loss due to death. Ithas now become clear that disturbances in apoptosis, also referred to asphysiological cell death or programmed cell death, that prevent or delaynormal cell turnover can be just as important to the pathogenesis ofdiseases as are known abnormalities in the regulation of proliferationand the cell cycle. Like cell division, which is controlled throughcomplex interactions between cell cycle regulatory proteins, apoptosisis similarly regulated under normal circumstances by the interaction ofgene products that either induce or inhibit cell death.

[0004] The stimuli which regulate the function of these apoptotic geneproducts include both extracellular and intracellular signals. Eitherthe presence or the removal of a particular stimuli can be sufficient toevoke a positive or negative apoptotic signal. For example,physiological stimuli that prevent or inhibit apoptosis include, forexample, growth factors, extracellular matrix, CD40 ligand, viral geneproducts, neutral amino acids, zinc, estrogen, and androgens. Incontrast, stimuli which promote apoptosis include growth factors such astumor necrosis factor (TNF), Fas, and transforming growth factor β(TGFβ), neurotransmitters, growth factor withdrawal, loss ofextracellular matrix attachment, intracellular calcium andglucocorticoids, for example.

[0005] Some of the well known regulators of apoptosis are cytokines ofthe tumor necrosis factor (TNF) ligand family, such as Fas ligand (FasL) and TNF, which induce apoptosis by activation of their correspondingreceptors, Fas and TNFR-1 (Nagata, S. (1997) Cell 88, 355-36S). Thesetwo receptors belong to a rapidly expanding family (collectively knownas the TNF-receptor family) containing at least eleven known members(Nagata, S. (1997) Cell 88, 355-365; Chinnaiyan, A. M. et al. (1997)Σχιενχε 6, 111-113). Members of this family contain an extracellularligand-binding domain, of 2-6 cysteine-rich repeats, which is about 25%conserved between different family members. The cytoplasmic region isless conserved between various members except for a stretch of about 80amino acids present in Fas, TNFR-1, DR3/Wsl-1/Apo-3/TRAMP, CAR-1 and DR4(Nagata, S. (1997) Cell 88, 355-365; and references therein). Thisintracellular region which has been designated the cytoplasmic “deathdomain” is responsible for transducing the death signal.

[0006] Activation of Fas results in recruitment of the Fas-associateddeath domain-containing molecule FADD/MORT-1, to the receptor complex(Boldin M. P. et al. (1995) J. Biol. Chem. 270, 7795-7798; Chinnaiyan A.M. et al. (1995) Cell 81, 505-512; Kischkel F. C. (1995) EMBO J. 14,5579-5588). The resulting signaling complex then triggers activation ofthe caspase apoptotic pathway through interaction of the N-terminaldeath effector domain (DED) of FADD with the corresponding motifs in theprodomain of caspase-8 (Mch5/MACH/FLICE) and probably caspase-10 (Mch4)(Boldin M. P. et al. (1996) Cell 85, 803-815; Bretz J. D. et al. (1996)Cell 85, 817-827; Alnemri E. S. et al. (1996) Proc. Natl. Sci. USA. 93,7464-7469; Alnemri E. S. et al. (1996) Cell 87,171.

[0007] In contrast to Fas, activation of TNFR-1 or DR3 results inrecruitment of another death domain-containing adaptor molecule known asTRAD (Chinnaiyan A. M. et al. (1996) Science 274, 990-991; Goeddel D. V.et al. (1995) Cell 81: 495-504). TRADD, can associate with a number ofsignaling molecules, including FADD, TRAF2, and RIP and as a result cantransduce an apoptotic signal as well as activate NF-KB (Goeddel D. V.et al. (1996) Cell 84, 299-308; Baichwal V. et al. (1996) Immunity 4,387-396). Consequently, engagement of TNFR-1 or DR3 can signal an arrayof diverse biological activities.

[0008] Recently, a new member of the TNF family known as TRAIL or Apo-2ligand was identified and shown to induce apoptosis in a variety oftumor cell lines (Davis T. D. et al. (1995) Immunity 3 673-682:Ashkenazi A. et al. (1996) J. Biol. Chem. 271, 12687-12690; Ashkenazi A.et al. (1996) Curr. Biol. 6, 750-752). However, it is unclear whatphysiological control mechanisms regulate this form of programmed celldeath or how the cell death pathways interact with other physiologicalprocesses within the organism.

[0009] Apoptosis functions in maintaining tissue homeostasis in a rangeof physiological processes such as embryonic development, immune cellregulation and normal cellular turnover. Therefore, the dysfunction, orloss of regulated apoptosis can lead to a variety of pathologicaldisease states. For example, the loss of apoptosis can lead to thepathological accumulation of self-reactive lymphocytes such as thatoccurring with many autoimmune diseases. Inappropriate loss of apoptosiscan also lead to the accumulation of virally infected cells and ofhyperproliferative cells such an neoplastic or tumor cells. Similarly,the inappropriate activation of apoptosis can also contribute to avariety of pathological disease states including, for example, acquiredimmunodeficiency syndrome (AIDS), neurodegenerative diseases andischemic injury. Treatments which are specifically designed to modulatethe apoptotic pathways in these and other pathological conditions canchange the natural progression of many of these diseases.

[0010] Thus, there exists a need to identify new apoptotic genes andtheir gene products and for methods of modulating this process for thetherapeutic treatment of human diseases. The present invention satisfiesthis need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0011] In accordance with the present invention, there are providednovel isolated mammalian members of the TRAIL-receptor family,designated DR5, TRAIL-R3, and splice variants thereof including DR5s.These invention proteins, or fragments thereof, are useful as immunogensfor producing anti-DR5 or anti-TRAIL-R3 antibodies, or in therapeuticcompositions containing such proteins and/or antibodies. The DR5 andTRAIL-R3 proteins are also useful in bioassays to identify agonists andantagonists thereto.

[0012] In accordance with the present invention, there are also providedisolated nucleic acids encoding novel DR5 or TRAIL-R3 proteins. Furtherprovided are vectors containing invention nucleic acids, probes thathybridize thereto, host cells transformed therewith, antisenseoligonucleotides thereto and related compositions. The nucleic acidmolecules described herein can be incorporated into a variety ofrecombinant expression systems known to those of skill in the art toreadily produce isolated recombinant DR5 or TRAIL-R3 proteins. Inaddition, the nucleic acid molecules of the present invention are usefulas probes for assaying for the presence and/or amount of a DR5 orTRAIL-R3 gene or mRNA transcript in a given sample. The nucleic acidmolecules described herein, and oligonucleotide fragments thereof, arealso useful as primers and/or templates in a PCR reaction for amplifyingnucleic acids encoding DR5 or TRAIL-R3 proteins. Also provided aretransgenic non-human mammals that express the invention proteins.

[0013] Antibodies that are immunoreactive with invention DR5 or TRAIL-R3proteins are also provided. These antibodies are useful in diagnosticassays to determine levels of DR5 or TRAIL-R3 proteins present in agiven sample, e.g., tissue samples, Western blots, and the like. Theantibodies can also be used to purify DR5 or TRAIL-R3 proteins fromcrude cell extracts and the like. Moreover, these antibodies areconsidered therapeutically useful to modulate the biological effect ofDR5 or TRAIL-R3 proteins in vivo.

[0014] Methods and diagnostic systems for determining the levels of DR5or TRAIL-R3 proteins in various tissue samples are also provided. Thesediagnostic methods can be used for monitoring the level oftherapeutically administered DR5 or TRAIL-R3 proteins or fragmentsthereof to facilitate the maintenance of therapeutically effectiveamounts. These diagnostic methods can also be used to diagnosephysiological disorders that result from abnormal levels or abnormalstructures of the DR5 or TRAIL-R3 proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1D illustrate sequence analysis and tissue distributionof DR5 and TRAIL-R3. Predicted amino acid sequence of human DR5 (A) andTRAIL-R3 (B). The mature DRS and TRAIL-R3 are predicted to start atGlu+1 and Tyr+1 (indicated by black diamonds), respectively. Theputative signal peptide and transmembrane domains are single- anddouble-underlined, respectively. The five identical repeats in theextracellular domain of TRAIL-R3 (B) are marked by black triangles. Theintracellular cytoplasmic death domain of DR5 (A) is boxed. C, Colinearalignment of the death domains of members of the TNF receptor family.Identical residues in at least three out of six sequences are shaded.The death domain of DR5 is 64, 30, 30, 20, 31% identical to thecorresponding domains in DR4, DR3, TNFR-1, Fas and CAR1 respectively.

[0016]FIGS. 2A and 2B illustrate that the extracellular domains of DR5and TRAIL-R3 bind TRAIL and can block TRAIL-induced apoptosis as setforth in Example III herein.

[0017] FIGS. 3A-3F illustrate that expression of DR5 but neitherTRAIL-R3 nor DR5s induces apoptosis in human cells as set forth inExample IV herein.

[0018]FIGS. 4A and 4B illustrate the in vivo interactions of DR5 as setforth in Example V herein.

[0019]FIG. 5 illustrates the nucleotide and predicted amino acidsequence of the splice variant DR5s.

[0020]FIG. 6 illustrates a colinear alignment of DR5s and DR5. Thedotted lines indicate sequences that are spliced in DR5 or DR5s.

DETAILED DESCRIPTION OF INVENTION

[0021] In accordance with the present invention, there are providednovel mammalian members of the TRAIL receptor protein family referred toherein as “DR5” and “TRAIL-R3”, and active fragments thereof. DR5 isalso referred to herein as “TRAIL-R2”. As used herein, the phrases “DR5”and “TRAIL-R3” refer to isolated and/or substantially pure mammalianproteins, preferably human, that are able to bind to the cytotoxicligand “TRAIL” (Wiley et al., 1995, Immunity, 3:673; and Marsters etal., 1996, Curr. Biol., 6:750), also known as Apo-2L. Invention DR5 andTRAIL-R3 proteins are further characterized by having the ability tomediate apoptosis. In their native environment, invention DR5 andTRAIL-R3 proteins are cell-surface receptor proteins.

[0022] Invention DR5 and TRAIL-R3 proteins include naturally occurringvariants thereof encoded by mRNA generated by alternative splicing of aprimary transcript (e.g., splice variant DR5s), and further includingactive fragments thereof which retain at least one native biologicalactivity, such as, for example, the immunogenic ability to generateanti-DR5 or anti-TRAIL-R3 antibodies, the ability to bind TRAIL ligand,the ability to modulate apoptosis, and the like. In isolated form,invention isolated DR5 and TRAIL-R3 proteins are free of cellularcomponents and/or contaminants normally associated with a native in vivoenvironment. As used herein, the term “polypeptide” refers to fulllength DR5 and TRAIL-R3 proteins or fragments thereof.

[0023] It has been found that DR5 but not TRAIL-R3, contains acytoplasmic “death domain” necessary for induction of apoptosis, andengages the apoptotic pathway independent of the adaptor moleculeFADD/Mort1. TRAIL-R3 on the other hand, can bind TRAIL but does notinduce apoptosis, and thus is contemplated to function as anantagonistic receptor. Similarly, DR5s contains a truncated death domainof DR5 and thus also functions as an antagonistic decoy receptor.

[0024] The invention DR5 proteins are further characterized by beingexpressed in at least the following cells: heart, brain, placenta, lung,liver, skeletal muscle, kidney and pancreas (see Figure ID and ExampleII). Other normal tissues such as testes, ovary, colon, small intestineand lymphoid tissues show detectable but low expression of DR5transcript. In addition, DR5 mRNA transcript was detected in thefollowing tumor cell lines: HL-60, promyelocytic leukemia; HeLa cell S3,K-562, chronic myelogenous leukemia; MOLT-4, lymphoblastic leukemia;Raji, Burkitt's lymphoma; SW480, colorectal adenocarcinoma; A549, lungcarcinoma; G361, melanoma (FIG. 1D). Surprisingly, it was found that theamount of DR5 transcript is at least 100-fold higher in most tumor celllines than in normal tissues. Thus, a correlation is contemplatedbetween the high sensitivity of tumor cells to TRAIL and the elevatedlevels of DR5 in these cells.

[0025] In addition, transient expression of DR5 triggers cytoplasmicdeath domain-dependent apoptosis (see FIGS. 3A and B). Similar to otherTNF-receptor family members, DR5-induced apoptosis is efficientlyblocked by the caspase inhibitors z-VAD-fmk and CrmA (FIG. 3D) and bythe dominant negative inhibitors FLAME-1, caspase-8-DN and caspase-10-DN(FIG. 3E). Thus, it is contemplated herein that the upstream caspases-8and -10 are involved in both the invention DR5 death signaling pathways.Moreover, unlike Fas, DR5 does not interact with the death-domaincontaining adaptor proteins FADD, CRADD, RIP or TRADD. Surprisingly,however, full length DR5, but not death domain-deleted mutants, arecapable of forming complexes with caspase-8, caspase-10 and FLAME-1.Because these proteins do not interact directly, it is likely that theformation of these DR5-related complexes requires an adaptor moleculedistinct from FADD.

[0026] In a particular embodiment of the present invention, an inventionDR5 is a protein of 411 amino acids (SEQ ID NO:2) with an overall ˜59%identity to DR4 (FIG. 1A). DR5's domain structure is highly related toDR4 and the other members of the TNF-receptor family. An invention DR5contains a putative N-terminal signal peptide (amino acids −51 to −1 ofFIG. 1A) followed by an extracellular domain containing twocysteine-rich pseudorepeats. Following the extracellular domain is atransmembrane domain (amino acids 132-152 of FIG. 1A) and a cytoplasmicdomain. Within the cytoplasmic domain there is a stretch of 67-aminoacids (amino acids 273-339 of FIG. 1A) comprising a death domainhomology region (FIG. 1C). The death domain of DR5 is 64, 30, 30, 20,31% identical to the corresponding domains in DR4, DR3, TNFR-1, Fas andCAR1, respectively. Based on these criteria and its apoptotic activity(see Example IV below) the novel TRAIL-R2 protein is also designateddeath receptor-5 (DR5).

[0027] In an additional embodiment of the present invention splicevariants are provided. Upon inspection of genomic clones of a TRAILreceptor, those skilled in the art are able to identify a variety ofpotential splice junctions. One such alternative splicing event yieldsDR5s which is an alternatively spliced isoform of DR5. DR5s is a proteinof 350 amino acids (SEQ ID NO:6) which is encoded by a cDNA of 1053nucleotides (SEQ ID NO:5). The mature DR5s protein contains acytoplasmic region with a truncated death domain (FIGS. 5 and 6) thatprovides a protein having functionality opposite to that of DR5. To thisend, DR5s substantially inhibits apoptosis by TRAIL, but not by TNF-α(FIG. 3F). Without wishing to be bound by theory, DR5s apparently actsas an inhibitory TRAIL-decoy receptor and thus may be able to protectcells against TRAIL-induced apoptosis.

[0028] The invention TRAIL-R3 proteins may be characterized as beingexpressed in at least the following cells: heart, brain, placenta,liver, skeletal muscle, kidney and pancreas (see FIG. 1D and ExampleII). In addition, TRAIL-R3 mRNA transcript was detected in the followingtumor cell lines: HeLa cell S3, K-562, chronic myelogenous leukemia;MOLT-4, lymphoblastic leukemia; Raji, Burkitt's lymphoma; SW480,colorectal adenocarcinoma; A549, lung carcinoma; G361, melanoma (FIG.1D). Surprisingly, relative to DR5 mRNA, a significantly elevatedexpression of TRAIL-R3 mRNA was observed in normal cells compared totumor cells.

[0029] In addition, it has been found that transient expression ofTRAIL-R3, which does not naturally contain a death domain, was incapableof inducing apoptosis (FIGS. 3A and B). Surprisingly, transientexpression of TRAIL-R3 in MCF7 cells significantly blocked TRAIL-inducedapoptosis (FIG. 3C), suggesting that TRAIL-R3 functions as anantagonistic decoy receptor.

[0030] Thus, since TRAIL-R3 does not contain a cytoplasmic death domain,and is capable of attenuating the cytotoxicity of TRAIL, TRAIL-R3 iscontemplated as functioning physiologically as an antagonist to DR4 andDR5.

[0031] In a particular embodiment of the present invention, an inventionTRAIL-R3 (SEQ ID NO:4) is a protein of 299 amino acids with an overall40 and 36% identity to DR4 and DR5, respectively (FIG. 1B). This proteincontains a putative N-terminal signal peptide (amino acids −63 to −1 ofFIG. 1B) followed by an extracellular domain containing twocysteine-rich pseudorepeats and five nearly identical PAAEETMN (T)TSPGTPA repeats (amino acids 139-153, 154-168, 169-183, 184-198, and199-213 of FIG. 1B, which corresponds to amino acids 202-216, 217-231,232-246, 247-261, and 262-276 of SEQ ID NO:4). Following theextracellular domain is a C-terminal transmembrane domain (amino acids217-236 of FIG. 1B). Unlike DR4 and DR5, this TRAIL-receptor does notcontain a cytoplasmic domain. Based on these criteria and its ability tobind TRAIL (see Example III below), this protein is designated TRAIL-R3.

[0032] As used herein, the term “apoptosis” refers to the well-knownprocess of programmed cell death. There is a variety of well-known,generally accepted in-vitro indicia of apoptosis, including nuclearmorphological changes, intemucleosomal fragmentation of DNA, theselective proteolysis of substrates, and the activation of CPP32-likecaspases. For example, the substrates of the caspase family of cysteineproteases have received considerable attention because cleavage of thesesubstrates offers molecular mechanisms for many of the hallmarkmorphological and functional changes exhibited by apoptotic cells(Casiano et al., J. Exp. Med. 184:765-770, (1996).

[0033] Use of the terms “isolated” and/or “purified” in the presentspecification and claims as a modifier of DNA, RNA, polypeptides orproteins means that the DNA, RNA, polypeptides or proteins so designatedhave been produced in such form by the hand of man, and thus areseparated from their native in vivo cellular environment. As a result ofthis human intervention, the recombinant DNAs, RNAs, polypeptides andproteins of the invention are useful in ways described herein that theDNAs, RNAs, polypeptides or proteins as they naturally occur are not.

[0034] As used herein, “mammalian” refers to the variety of species fromwhich invention DR5 or TRAIL-R3 proteins are derived, e.g. human, rat,mouse, rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline,and the like.

[0035] Presently preferred DR5 and TRAIL-R3 proteins of the inventioninclude amino acid sequences that are substantially the same as theprotein sequence set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively,as well as biologically active, modified forms thereof. Those of skillin the art will recognize that numerous residues of the above-describedsequences can be substituted with other, chemically, sterically and/orelectronically similar residues without substantially altering thebiological activity of the resulting protein species. In addition,larger or smaller polypeptide sequences containing substantially thesame sequence as SEQ ID NO:2 or SEQ ID NO:4 therein (e.g., splicevariants including but not limited to SEQ ID NO:6, active fragments ofAT, and the like) are contemplated.

[0036] As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95%, about97%, about 99%, up to 100% amino acid sequence identity being especiallypreferred. Such amino acid sequence identity measurements may bedetermined by standard methodologies, including use of the NationalCenter for Biotechnology Information BLAST search methodology availableat www.ncbi.nlm.nih.gov. The identity methodologies most preferred arethose described in U.S. Pat. No. 5,691,179 and Altschul et al., NucleicAcids Res. 25:3389-3402, 1997, both of which are incorporated herein byreference. It is recognized, however, that DR5 and TRAIL-R3 proteinsarising as splice variants (or DR5 and TRAIL-R3 nucleic acids referredto herein) containing less than the described levels of sequenceidentity or that are modified by conservative amino acid substitutions,or by substitution of degenerate codons, are also encompassed within thescope of the present invention. A preferred DR5 protein disclosedherein, is human DR5 set forth as SEQ ID NO:2. A preferred TRAIL-R3protein disclosed herein, is human TRAIL-R3 set forth as SEQ ID NO:4. Apreferred DR5 splice variant protein disclosed herein, is human DR5s,set forth as SEQ ID NO:6.

[0037] The term “biologically active” or “functional”, when used hereinas a modifier of invention DR5 and TRAIL-R3 proteins, or polypeptidefragment thereof, refers to a polypeptide that exhibits at least one ofthe functional characteristics attributed to TRAIL receptors. Forexample, one biological activity of invention DR5 and TRAIL-R3 proteinsis the ability to bind to the TRAIL ligand. Other biological activitiesof invention DR5 and TRAIL-R3 proteins include the ability to modulateapoptosis (i.e., increasing or decreasing the level of apoptosis), theability to bind intracellular adaptor proteins, and the like.

[0038] Yet another biological activity of invention DR5 or TRAIL-R3proteins is the ability to act as an immunogen for the production ofpolyclonal and monoclonal antibodies that bind specifically to DR5 orTRAIL-R3. Thus, an invention nucleic acid encoding DR5 or TRAIL-R3 willencode a polypeptide specifically recognized by an antibody that alsospecifically recognizes the DR5 or TRAIL-R3 proteins (preferably human)including the sequences set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQID NO:6. Such activity may be assayed by any method known to those ofskill in the art. For example, a test polypeptide encoded by a DR5 orTRAIL-R3 cDNA can be used to produce antibodies, which may then beassayed for their ability to bind to the protein including the sequenceset forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. If the antibodybinds to the test-polypeptide and the protein including the sequence setforth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 with substantially thesame affinity, then the polypeptide possesses the required biologicalactivity.

[0039] The invention DR5 or TRAIL-R3 proteins can be isolated by avariety of methods well-known in the art, e.g. the recombinantexpression systems described herein, precipitation, gel filtration,ion-exchange, reverse-phase and affinity chromatography, and the like.Other well-known methods are described in Deutscher et al., Guide toProtein Purification: Methods in Enzymology Vol. 182, (Academic Press,(1990)), which is incorporated herein by reference. Alternatively, theisolated polypeptides of the present invention can be obtained usingwell-known recombinant methods as described, for example, in Sambrook etal., (supra., 1989).

[0040] An example of the means for preparing the inventionpolypeptide(s) is to express nucleic acids encoding the DR5 or TRAIL-R3in a suitable host cell, such as a bacterial cell, a yeast cell, anamphibian cell (i.e., oocyte), or a mammalian cell, using methods wellknown in the art, and recovering the expressed polypeptide, again usingwell-known methods. Invention DR5 or TRAIL-R3 proteins may be isolateddirectly from cells that have been transformed with expression vectorsas described herein. The invention polypeptide, biologically activefragments, and functional equivalents thereof can also be produced bychemical synthesis. For example, synthetic polypeptides can be producedusing Applied Biosystems, Inc. Model 430A or 431A automatic peptidesynthesizer (Foster City, Calif.) employing the chemistry provided bythe manufacturer.

[0041] Also encompassed by the term DR5 or TRAIL-R3 are active fragmentsor polypeptide analogs thereof. The term “active fragment” refers to apeptide fragment that is a portion of a full length DR5 or TRAIL-R3protein, provided that the portion has a biological activity, as definedabove, that is characteristic of at least one function of thecorresponding full length protein. For example, an active fragment of anDR5 or TRAIL-R3 protein, such as an extracellular domain can have anactivity such as the ability, for example, to bind TRAIL ligand or tomodulate the level of apoptosis after binding to TRAIL. Thecharacteristic of an active fragment of invention DR5 or TRAIL-R3proteins to elicit an immune response is useful for obtaining ananti-TRAIL receptor antibody. Thus, the invention also provides activefragments of invention DR5 and TRAIL-R3 proteins, which can beidentified using the binding and bioassays described herein.

[0042] The term “polypeptide analog” includes any polypeptide having anamino acid residue sequence substantially identical to a sequencespecifically shown herein in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the ability to mimic DR5 or TRAIL-R3 as described herein.Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another.

[0043] The amino acid length of active fragments or polypeptide analogsof the present can range from about 5 amino acids up to the full-lengthprotein sequence of DR5 or TRAIL-R3. In certain embodiments, the aminoacid lengths include, for example, at least about 10 amino acids, atleast about 20, at least about 30, at least about 40; at least about 50,at least about 75, at least about 100, at least about 150, at leastabout 200, at least about 250 or more amino acids in length up to thefull-length DR5 or TRAIL-R3 protein sequence.

[0044] As used herein the phrase “conservative substitution” alsoincludes the use of a chemically derivatized residue in place of anon-derivatized residue, provided that such polypeptide displays therequired binding activity. The phrase “chemical derivative” refers to asubject polypeptide having one or more residues chemically derivatizedby reaction of a functional side group. Such derivatized moleculesinclude, for example, those molecules in which free amino groups havebeen derivatized to form amine hydrochlorides, p-toluene sulfonylgroups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetylgroups or formyl groups. Free carboxyl groups may be derivatized to formsalts, methyl and ethyl esters or other types of esters or hydrazides.Free hydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine may be derivatized toform N-im-benzylhistidine. Also included as chemical derivatives arethose peptides which contain one or more naturally occurring amino acidderivatives of the twenty standard amino acids. For examples:4-hydroxyproline may be substituted for proline; S-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and omithine may besubstituted for lysine. Polypeptides of the present invention alsoinclude any polypeptide having one or more additions and/or deletions ofresidues, relative to the sequence of a polypeptide whose sequence isshown herein, so long as the required activity is maintained.

[0045] The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified DR5 or TRAIL-R3polypeptide, an active fragment or polypeptide analog thereof, or apurified, mature protein and active fragments thereof, alone or incombination with each other. These polypeptides or proteins can berecombinantly derived, chemically synthesized or purified from nativesources. As used herein, the term “acceptable carrier” encompasses anyof the standard pharmaceutical carriers, such as phosphate bufferedsaline solution, water and emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents.

[0046] In accordance with another embodiment of the present invention,there are provided isolated nucleic acids, which encode invention DR5 orTRAIL-R3 proteins, and fragments thereof. The nucleic acid moleculesdescribed herein are useful for producing invention proteins, when suchnucleic acids are incorporated into a variety of protein expressionsystems known to those of skill in the art. In addition, such nucleicacid molecules or fragments thereof can be labeled with a readilydetectable substituent for use as hybridization probes to assay for thepresence and/or amount of an DR5 or TRAIL-R3 gene or mRNA transcript ina given sample. The nucleic acid molecules described herein, andfragments thereof, are also useful as primers and/or templates in a PCRreaction for amplifying genes encoding the invention protein describedherein.

[0047] The term “nucleic acid” (also referred to as polynucleotides)encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),probes, oligonucleotides, and primers. DNA can be either complementaryDNA (cDNA) or genomic DNA, e.g. a gene encoding an DRS or TRAIL-R3protein. One means of isolating a nucleic acid encoding a DR5 orTRAIL-R3 polypeptide is to probe a mammalian genomic or cDNA librarywith a natural or artificially designed DNA probe using methods wellknown in the art. DNA probes derived from nucleic acid encoding DRS orTRAIL-R3 proteins are particularly useful for this purpose. DNA and cDNAmolecules that encode DR5 or TRAIL-R3 proteins can be used to obtaincomplementary genomic DNA, cDNA or RNA from mammalian (e.g., human,mouse, rat, rabbit, pig, and the like), or other animal sources, or toisolate related cDNA or genomic clones by the screening of cDNA orgenomic libraries, by methods described in more detail below. Suchnucleic acids may include, but are not limited to, nucleic acids havingsubstantially the same nucleotide sequence as set forth in SEQ ID NO:1or SEQ ID NO:3, or splice variant cDNA sequences thereof.

[0048] Also encompassed by the terms DR5 or TRAIL-R3 may be “splicevariant” or “alternatively spliced” proteins thereof. These terms areused herein to describe a particular nucleotide sequence encoding aninvention receptor and refers to a cDNA sequence or protein encodedthereby that results from the well known eukaryotic RNA splicingprocess. The RNA splicing process may involve the removal of introns andthe joining of exons from eukaryotic primary RNA transcripts to createmature RNA molecules of the cytoplasm. Methods of isolating splicevariant nucleotide sequences are well known in the art. For example, oneskilled in the art can employ nucleotide probes derived from the DR5 orTRAIL-R3 encoding cDNA of SEQ ID NO: 1 or SEQ ID NO:3 to screen a cDNAor genomic library as described herein. A preferred splice variant isthe alternatively spliced isoform of DR5, or DR5short (DR5s). The cDNAencoding this splice variant is set forth in SEQ ID NO:5.

[0049] Alternative splicing may play an important role in regulation ofapoptosis. For example, alternative splicing of the Bcl-x, Ced-4, andIch-1 pre-mRNA produces products that play opposite roles in apoptosis.In this regard, as described herein, DR5s may be similar in that thisisoform may be capable of inhibiting apoptosis while the larger form(DR5) may promote apoptosis (see FIG. 3F).

[0050] In one embodiment of the present invention, cDNAs encoding theinvention DR5 and TRAIL-R3 proteins disclosed herein includesubstantially the same nucleotide sequence as set forth in SEQ ID NO: 1,SEQ ID NO:3, and SEQ ID NO:5.

[0051] As employed herein, the term “substantially the same nucleotidesequence” refers to DNA having sufficient identity to the referencepolynucleotide, such that it hybridizes to the reference nucleotideunder moderately stringent hybridization conditions. In one embodiment,DNA having substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 or alarger amino acid sequence including SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:6. In another embodiment, DNA having “substantially the samenucleotide sequence” as the reference nucleotide sequence has at least60% identity with respect to the reference nucleotide sequence. DNAhaving at least 70%, at least 80%, more preferably at least 90%, yetmore preferably at least 95%, with up to at least 97%, and at least 99%identity to the reference nucleotide sequence is preferred.

[0052] The present invention also encompasses nucleic acids that differfrom the nucleic acids shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:5, but which have the same phenotype. Phenotypically similar nucleicacids are also referred to as “functionally equivalent nucleic acids”.As used herein, the phrase “functionally equivalent nucleic acids”encompasses nucleic acids characterized by slight and non-consequentialsequence variations that function in substantially the same manner toproduce the same protein product(s) as the nucleic acids disclosedherein. In particular, functionally equivalent nucleic acids may encodepolypeptides that are the same as those disclosed herein or that haveconservative amino acid variations, or that encode larger polypeptidesthat include SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. For example,conservative variations include substitution of a non-polar residue withanother non-polar residue, or substitution of a charged residue with asimilarly charged residue. These variations include those recognized byskilled artisans as those that do not substantially alter the tertiarystructure of the protein.

[0053] Further provided are nucleic acids encoding DR5 and TRAIL-R3proteins that, by virtue of the degeneracy of the genetic code, do notnecessarily hybridize to the invention nucleic acids under specifiedhybridization conditions. Preferred nucleic acids encoding the inventionDR5 or TRAIL-R3 polypeptide are comprised of nucleotides that encodesubstantially the same amino acid sequence set forth in SEQ ID NO:2, SEQID NO:4, or splice variants thereof, respectively.

[0054] Thus, an exemplary nucleic acid encoding an invention DR5 orTRAIL-R3 protein may be selected from:

[0055] (a) DNA encoding the amino acid sequence set forth in SEQ ID NO:2or SEQ ID NO:4;

[0056] (b) DNA that hybridizes to the DNA of (a) under moderatelystringent conditions, wherein said DNA encodes biologically active DR5or TRAIL-R3; or

[0057] (c) DNA degenerate with respect to either (a) or (b) above,wherein said DNA encodes biologically active DR5 or TRAIL-R3.

[0058] Hybridization refers to the binding of complementary strands ofnucleic acid (i.e., sense:antisense strands or probe:target-DNA) to eachother through hydrogen bonds, similar to the bonds that naturally occurin chromosomal DNA. Stringency levels used to hybridize a given probewith target-DNA can be readily varied by those of skill in the art.

[0059] The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose skilled in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

[0060] As used herein, the phrase “moderately stringent hybridization”refers to conditions that permit target-DNA to bind a complementarynucleic acid that has about 60% identity, preferably about 75% identity,more preferably about 85% identity to the target DNA; with greater thanabout 90% identity to target-DNA being especially preferred. Preferably,moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5× Denhart's solution, 5× SSPE, 0.2% SDSat 42° C., followed by washing in 0.5× SSPE, 0.2% SDS, at 42° C.

[0061] The phrase “high stringency hybridization” refers to conditionsthat permit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stablein 0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein). High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5× Denhart'ssolution, 5× SSPE, 0.2% SDS at 42° C., followed by washing in 0.1× SSPE,and 0.1% SDS at 65° C.

[0062] The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5× Denhart's solution, 6×SSPE, 0.2% SDS at 37° C., followed by washing in 1× SSPE, 0.2% SDS, at50° C. Denhart's solution and SSPE (see, e.g., Sambrook et al.,Molecular Cloning. A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989) are well known to those of skill in the art as are othersuitable hybridization buffers.

[0063] As used herein, the term “degenerate” refers to codons thatdiffer in at least one nucleotide from a reference nucleic acid, e.g.,SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, but encode the same aminoacids as the reference nucleic acid. For example, codons specified bythe triplets “UCU”, “UCC”, “UCA”, and “UCG” are degenerate with respectto each other since all four of these codons encode the amino acidserine.

[0064] Preferred nucleic acids encoding the invention polypeptide(s)hybridize under moderately stringent, preferably high stringency,conditions to substantially the entire sequence of the nucleic acidsequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0065] Site-directed mutagenesis of any region of DR5 or TRAIL-R3 cDNAis contemplated herein for the production of mutant DR5 or TRAIL-R3cDNAs. For example, the Transformer Mutagenesis Kit (available fromClontech, Palo Alto, Calif.) can be used to construct a variety ofmissense and/or nonsense mutations to DR5 or TRAIL-R3 cDNA, and thelike.

[0066] The invention nucleic acids may be produced by a variety ofmethods well-known in the art, e.g., the methods described herein,employing PCR amplification using oligonucleotide primers from variousregions of SEQ ID NO: 1 or SEQ ID NO:3, and the like.

[0067] In accordance with a further embodiment of the present invention,optionally labeled DR5-encoding and TRAIL-R3-encoding cDNAs, orfragments thereof, can be employed to probe library(ies) (e.g., cDNA,genomic, and the like) for additional nucleic acid sequences encodingrelated novel mammalian DR5 and TRAIL-R3 proteins. Construction ofmammalian cDNA and genomic libraries, preferably a human library, iswell-known in the art.

[0068] Screening of such a cDNA or genomic library is initially carriedout under low-stringency conditions, which comprise a temperature ofless than about 42° C., a formamide concentration of less than about50%, and a moderate to low salt concentration.

[0069] In one embodiment, probe-based screening conditions comprise atemperature of about 37° C., a formamide concentration of about 20%, anda salt concentration of about 5× standard saline citrate (SSC; 20× SSCcontains 3M sodium chloride, 0.3M sodium citrate, pH 7.01). Suchconditions will allow the identification of sequences which have asubstantial degree of similarity with the probe sequence, withoutrequiring perfect homology. The phrase “substantial similarity” refersto sequences which share at least 50% homology. Preferably,hybridization conditions will be selected which allow the identificationof sequences having at least 70% homology with the probe, whilediscriminating against sequences which have a lower degree of homologywith the probe. As a result, nucleic acids having substantially the same(i.e., similar) nucleotide sequence as SEQ ID NO:1, SEQ ID NO:3, orsplice variants thereof including SEQ ID NO:5, are obtained.

[0070] As used herein, a nucleic acid “oligonucleotide”, also referredto herein as a probe or primer, is single-stranded DNA or RNA, oranalogs thereof, that has a sequence of nucleotides that includes, forexample, at least 14, at least 20, at least 50, at least 100, at least200, at least 300, at least 400, or at least 500 contiguous nucleotidebases that are the same as (or the complement of) any contiguous basesset forth in any of SEQ ID NO:1 or SEQ ID NO:3. Preferred regions fromwhich to construct probes include 5′ and/or 3′ coding regions of SEQ IDNO:1, SEQ ID NO:3, or splice variants thereof. In addition, the entirecDNA encoding region of an invention protein, such as the entiresequence corresponding to SEQ ID NO:1 (DR5) or SEQ ID NO:3 (TRAIL-R3),may be used as a probe. Further, when probing a splice variant, thesplice junctions or intervening sequences may be used to construct aprobe (see FIG. 6). Probes may be labeled by methods well-known in theart, as described hereinafter, and used in various diagnostic kits.

[0071] As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to invention nucleicacid probes, expressed proteins, polypeptide fragments, or antibodymolecules. These atoms or molecules can be used alone or in conjunctionwith additional reagents. Such labels are themselves well-known inclinical diagnostic chemistry.

[0072] Also provided are antisense oligonucleotides having a sequencecapable of binding specifically with any portion of an mRNA that encodesDR5 or TRAIL-R3 proteins so as to prevent translation of the mRNA. Theantisense oligonucleotide may have a sequence capable of bindingspecifically with any portion of the sequence of the cDNA encoding DR5or TRAIL-R3 proteins. As used herein, the phrase “binding specifically”encompasses the ability of a nucleic acid sequence to recognize acomplementary nucleic acid sequence and to form double-helical segmentstherewith via the formation of hydrogen bonds between the complementarybase pairs. An example of an antisense oligonucleotide is an antisenseoligonucleotide comprising chemical analogs of nucleotides.

[0073] Compositions comprising an amount of the antisenseoligonucleotide, described above, effective to reduce expression of DR5or TRAIL-R3 proteins by passing through a cell membrane and bindingspecifically with mRNA encoding DR5 or TRAIL-R3 proteins so as toprevent translation and an acceptable hydrophobic carrier capable ofpassing through a cell membrane are also provided herein. Suitablehydrophobic carriers are described, for example, in U.S. Pat. Nos.5,334,761; 4,889,953; 4,897,355, and the like. The acceptablehydrophobic carrier capable of passing through cell membranes may alsocomprise a structure which binds to a receptor specific for a selectedcell type and is thereby taken up by cells of the selected cell type.The structure may be part of a protein known to bind to a cell-typespecific receptor, e.g., FGF and other growth factors.

[0074] Antisense oligonucleotide compositions are useful to inhibittranslation of mRNA encoding invention polypeptides. Syntheticoligonucleotides, or other antisense chemical structures are designed tobind to mRNA encoding DR5 or TRAIL-R3 proteins and inhibit translationof mRNA and are useful as compositions to inhibit expression of DR5 orTRAIL-R3 associated genes in a tissue sample or in a subject.

[0075] In accordance with another embodiment of the invention, kits areprovided for detecting the presence of a DR5 or TRAIL-R3 nucleicsequence comprising at least one oligonucleotide, e.g., a probe orantisense oligonucleotide, according to the present invention. Such kitscan be used for detecting mutations, duplications, deletions, splicevariant transcripts, rearrangements or aneuploidies in a DR5 or TRAIL-R3gene.

[0076] The present invention provides means to modulate levels ofexpression of DR5 or TRAIL-R3 proteins by employing synthetic antisenseoligonucleotide compositions (hereinafter SAOC) which inhibittranslation of mRNA encoding these polypeptides. Syntheticoligonucleotides, or other antisense chemical structures designed torecognize and selectively bind to mRNA, are constructed to becomplementary to portions of the DR5 or TRAIL-R3 coding strand ornucleotide sequences shown in SEQ ID NO:1 or SEQ ID NO:3. The SAOC isdesigned to be stable in the blood stream for administration to asubject by injection or by direct tumor site integration, or stable inlaboratory cell culture conditions. The SAOC is designed to be capableof passing through the cell membrane in order to enter the cytoplasm ofthe cell by virtue of physical and chemical properties of the SAOC whichrender it capable of passing through cell membranes, for example, bydesigning small, hydrophobic SAOC chemical structures, or by virtue ofspecific transport systems in the cell which recognize and transport theSAOC into the cell. In addition, the SAOC can be designed foradministration only to certain selected cell populations by targetingthe SAOC to be recognized by specific cellular uptake mechanisms whichbind and take up the SAOC only within select cell populations.

[0077] For example, the SAOC may be designed to bind to a receptor foundonly in a certain cell type, as discussed supra. The SAOC is alsodesigned to recognize and selectively bind to target mRNA sequence,which may correspond to a sequence contained within the sequence shownin SEQ ID NO:1, SEQ ID NO:3, or splice variants thereof. The SAOC isdesigned to inactivate target mRNA sequence by either binding theretoand inducing degradation of the mRNA by, for example, RNase I digestion,or inhibiting translation of mRNA target sequence by interfering withthe binding of translation-regulating factors or ribosomes, or inclusionof other chemical structures, such as ribozyme sequences or reactivechemical groups which either degrade or chemically modify the targetmRNA. SAOCs have been shown to be capable of such properties whendirected against mRNA targets (see Cohen et al., TIPS, 10:435 (1989) andWeintraub, Sci. American, January (1990), p.40; both incorporated hereinby reference).

[0078] In accordance with yet another embodiment of the presentinvention, there is provided a method for the recombinant production ofinvention DR5 or TRAIL-R3 proteins by expressing the above-describednucleic acid sequences in suitable host cells. Recombinant DNAexpression systems that are suitable to produce DR5 and TRAIL-R3proteins described herein are well-known in the art. For example, theabove-described nucleotide sequences can be incorporated into vectorsfor further manipulation. As used herein, vector (or plasmid) refers todiscrete elements that are used to introduce heterologous DNA into cellsfor either expression or replication thereof.

[0079] Suitable expression vectors are well-known in the art, andinclude vectors capable of expressing DNA operatively linked to aregulatory sequence, such as a promoter region that is capable ofregulating expression of such DNA. Thus, an expression vector refers toa recombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the inserted DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome. In addition, vectors may containappropriate packaging signals that enable the vector to be packaged by anumber of viral virions, e.g., retroviruses, herpes viruses,adenoviruses, resulting in the formation of a “viral vector.”

[0080] As used herein, the term “operatively linked” refers to thefunctional relationship of DNA with regulatory and effector nucleotidesequences, such as promoters, enhancers, transcriptional andtranslational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA.

[0081] Suitable transformation vectors are well-known in the art andinclude Blueskript and phage Lambda ZAP vectors (Stratagene, La Jolla,Calif.), and the like. Other suitable vectors and promoters aredisclosed in detail in U.S. Pat. No. 4,798,885, issued Jan. 17, 1989,the disclosure of which is incorporated herein by reference in itsentirety.

[0082] In accordance with another embodiment of the present invention,there are provided “recombinant cells” containing the nucleic acidmolecules (i.e., DNA or mRNA) of the present invention. Methods oftransforming suitable host cells, preferably bacterial cells, and morepreferably E. coli cells, as well as methods applicable for culturingsaid cells containing a gene encoding a heterologous protein, aregenerally known in the art. See, for example, Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989).

[0083] Exemplary methods of introducing (transducing) expression vectorscontaining invention nucleic acids into host cells to produce transducedrecombinant cells (i.e., cells containing recombinant heterologousnucleic acid) are well-known in the art (see, for review, Friedmann,Science, 244:1275-1281 (1989); Mulligan, Science, 260:926-932 (1993),each of which are incorporated herein by reference in their entirety).Exemplary methods of transduction include, e.g., infection employingviral vectors (see, e.g., U.S. Pat. No. 4,405,712 and 4,650,764),calcium phosphate transfection (U.S. Pat. Nos. 4,399,216 and 4,634,665),dextran sulfate transfection, electroporation, lipofection (see, e.g.,U.S. Pat. Nos. 4,394,448 and 4,619,794), cytofection, particle beadbombardment, and the like. The heterologous nucleic acid can optionallyinclude sequences which allow for its extrachromosomal (i.e., episomal)maintenance, or the heterologous DNA can be caused to integrate into thegenome of the host (as an alternative means to ensure stable maintenancein the host).

[0084] Host organisms contemplated for use in the practice of thepresent invention include those organisms in which recombinantproduction of heterologous proteins has been carried out. Examples ofsuch host organisms include bacteria (e.g., E. coli), yeast (e.g.,Saccharomyces cerevisiae, Candida tropicalis, Hansenula polymorpha andP. pastoris; see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555and 4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk- cells),insect cells, and the like.

[0085] In one embodiment, nucleic acids encoding the invention DR5 orTRAIL-R3 proteins can be delivered into mammalian cells, either in vivoor in vitro using suitable viral vectors well-known in the art (e.g.,retroviral vectors, adenovirus vectors, and the like). In addition,where it is desirable to limit or reduce the in vivo expression of theinvention DR5 or TRAIL-R3 proteins, the introduction of the antisensestrand of the invention nucleic acid is contemplated.

[0086] Viral based systems provide the advantage of being able tointroduce relatively high levels of the heterologous nucleic acid into avariety of cells. Suitable viral vectors for introducing inventionnucleic acid encoding an DR5 or TRAIL-R3 protein into mammalian cells(e.g., vascular tissue segments) are well known in the art. These viralvectors include, for example, Herpes simplex virus vectors (e.g., Gelleret al., Science, 241:1667-1669 (1988)), Vaccinia virus vectors (e.g.,Piccini et al., Meth. in Enzymology, 153:545-563 (1987); Cytomegalovirusvectors (Mocarski et al., in Viral Vectors, Y. Gluzman and S.H. Hughes,Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp.78-84), Moloney murine leukemia virus vectors (Danos et al., PNAS USA,85:6469 (1980)), adenovirus vectors, (e.g., Logan et al., PNAS USA,81:3655-3659 (1984); Jones et al., Cell, 17:683-689 (1979); Berkner,Biotechniques, 6:616-626 (1988); Cotten et al., PNAS, USA, 89:6094-6098(1992); Graham et al., Meth. Mol. Biol, 7:109-127 (1991)),adeno-associated virus vectors, retrovirus vectors (see, e.g., U.S. Pat.Nos. 4,405,712 and 4,650,764), and the like. Especially preferred viralvectors are the adenovirus and retroviral vectors.

[0087] For example, in one embodiment of the present invention,adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes(Wagner et al., PNAS. USA, 89:6099-6103 (1992); Curiel et al., Hum. GeneThera v, 3:147-154 (1992); Gao et al., Hum. Gene Ther., 4:14-24 (1993))are employed to transduce mammalian cells with heterologous DR5 orTRAIL-R3 nucleic acid. Any of the plasmid expression vectors describedherein may be employed in a TfAdpl-DNA complex.

[0088] As used herein, “retroviral vector” refers to the well-known genetransfer plasmids that have an expression cassette encoding anheterologous gene residing between two retroviral LTRs. Retroviralvectors typically contain appropriate packaging signals that enable theretroviral vector, or RNA transcribed using the retroviral vector as atemplate, to be packaged into a viral virion in an appropriate packagingcell line (see, e.g., U.S. Pat. No. 4,650,764).

[0089] Suitable retroviral vectors for use herein are described, forexample, in U.S. Pat. No. 5,252,479, and in WIPO publications WO92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829,incorporated herein by reference, which provide a description of methodsfor efficiently introducing nucleic acids into human cells using suchretroviral vectors. Other retroviral vectors include, for example, themouse mammary tumor virus vectors (e.g., Shackleford et al., PNAS, USA,85:9655-9659 (1988)), and the like.

[0090] In accordance with yet another embodiment of the presentinvention, there are provided anti-DR5 and anti -TRAIL-R3 antibodieshaving specific reactivity with DR5 and TRAIL-R3 proteins, respectively,of the present invention. Active fragments of antibodies are encompassedwithin the definition of “antibody”. Invention antibodies can beproduced by methods known in the art using invention DR5 or TRAIL-R3proteins, or portions thereof as antigens. For example, polyclonal andmonoclonal antibodies can be produced by methods well known in the art,as described, for example, in Harlow and Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory (1988)), which is incorporatedherein by reference.

[0091] Invention DR5 and TRAIL-R3 proteins can be used as immunogens ingenerating such antibodies. Alternatively, synthetic peptides can beprepared (using commercially available synthesizers) and used asimmunogens. Amino acid sequences can be analyzed by methods well knownin the art to determine whether they encode hydrophobic or hydrophilicdomains of the corresponding polypeptide. Altered antibodies such aschimeric, humanized, CDR-grafted or bifunctional antibodies can also beproduced by methods well known in the art. Such antibodies can also beproduced by hybridoma, chemical synthesis or recombinant methodsdescribed, for example, in Sambrook et al., supra., and Harlow and Lane,supra. Both anti-peptide and anti-fusion protein antibodies can be used.(see, for example, Bahouth et al., Trends Pharmacol. Sci. 12:338 (1993);Ausubel et al., Current Protocols in Molecular Biology (John Wiley andSons, NY (1989) which are incorporated herein by reference).

[0092] Antibody so produced can be used, inter alia, in diagnosticmethods and systems to detect the level of DR5 or TRAIL-R3 proteinpresent in a mammalian, preferably human, body sample, such as tissue orvascular fluid. Such antibodies can also be used for the immunoaffinityor affinity chromatography purification of the invention DR5 andTRAIL-R3 proteins. In addition, methods are contemplated herein fordetecting the presence of DR5 or TRAIL-R3 proteins either on the surfaceof a cell or within a cell, which methods comprise contacting the cellwith an antibody that specifically binds to DR5 or TRAIL-R3 proteins,under conditions permitting binding of the antibody to DR5 or TRAIL-R3proteins, detecting the presence of the antibody bound to DR5 orTRAIL-R3, and thereby detecting the presence of invention polypeptideson the surface of, or within, the cell. With respect to the detection ofsuch polypeptides, the antibodies can be used for in vitro diagnostic orin vivo imaging methods.

[0093] Immunological procedures useful for in vitro detection of targetDR5 or TRAIL-R3 proteins in a sample include immunoassays that employ adetectable antibody. Such immunoassays include, for example, ELISA,Pandex microfluorimetric assay, agglutination assays, flow cytometry,serum diagnostic assays and immunohistochemical staining procedureswhich are well known in the art. An antibody can be made detectable byvarious means well known in the art. For example, a detectable markercan be directly or indirectly attached to the antibody. Useful markersinclude, for example, radionucleotides, enzymes, fluorogens, chromogensand chemiluminescent labels.

[0094] Invention anti-DR5 or TRAIL-R3 antibodies are contemplated foruse herein to modulate activity of the DR5 or TRAIL-R3 polypeptide inliving animals, in humans, or in biological tissues or fluids isolatedtherefrom. The term “modulate” refers to a compound's ability toincrease (e.g., via an agonist) or inhibit (e.g., via an antagonist) thebiological activity of DR5 or TRAIL-R3 protein, such as the apoptosismediating activity of DR5 or TRAIL-R3. Accordingly, compositionscomprising a carrier and an amount of an antibody having specificity forDR5 or TRAIL-R3 proteins effective to block naturally occurring ligands,such as TRAIL, or other DR5-binding or TRAIL-binding proteins frombinding to invention DR5 or TRAIL-R3 proteins are contemplated herein.For example, a monoclonal antibody directed to an epitope of DR5 orTRAIL-R3 present on the surface of a cell that has an amino acidsequence substantially the same as an amino acid sequence shown in SEQID NO:2, SEQ ID NO:4, or splice variant thereof (e.g., SEQ ID NO:6), canbe useful for this purpose.

[0095] The present invention further provides transgenic non-humanmammals that are capable of expressing exagenous nucleic acids encodingDR5 or TRAIL-R3 proteins. As employed herein, the phrase “exagenousnucleic acid” refer to nucleic acid sequence which is not native to thehost, or which is present in the host in other than its nativeenvironment (e.g., as part of a genetically engineered DNA construct).In addition to naturally occurring levels of invention TRAIL receptors,invention DR5 or TRAIL-R3 proteins can either be overexpressed,underexpressed, or expressed in an inactive mutated form (such as in thewell-known knockout transgenics) in transgenic mammals.

[0096] Also provided are transgenic non-human mammals capable ofexpressing nucleic acids encoding DR5 or TRAIL-R3 proteins so mutated asto be incapable of normal activity, i.e., do not express native DR5 orTRAIL-R3. The present invention also provides transgenic non-humanmammals having a genome comprising antisense nucleic acids complementaryto nucleic acids encoding DR5 or TRAIL-R3 proteins, placed so as to betranscribed into antisense mRNA complementary to mRNA encoding DR5 orTRAIL-R3 proteins, which hybridizes to the mRNA and, thereby, reducesthe translation thereof. The nucleic acid may additionally comprise aninducible promoter and/or tissue specific regulatory elements, so thatexpression can be induced, or restricted to specific cell types.Examples of nucleic acids are DNA or cDNA having a coding sequencesubstantially the same as the coding sequence shown in SEQ ID NO: 1, SEQID NO:3, or SEQ ID NO:5. An example of a non-human transgenic mammal isa transgenic mouse.

[0097] Animal model systems which elucidate the physiological andbehavioral roles of DR5 or TRAIL-R3 proteins are also provided, and areproduced by creating transgenic animals in which the expression of theDR5 or TRAIL-R3 polypeptide is altered using a variety of techniques.Examples of such techniques include the insertion of normal or mutantversions of nucleic acids encoding a DR5 or TRAIL-R3 polypeptide bymicroinjection, retroviral infection or other means well known to thoseskilled in the art, into appropriate fertilized embryos to produce atransgenic animal. (See, for example, Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory,(1986)).

[0098] Also contemplated herein, is the use of homologous recombinationof mutant or normal versions of genes encoding DR5 or TRAIL-R3 proteinswith the native gene locus in transgenic animals, to alter theregulation of expression or the structure of (see, Capecchi et al.,Science, 244:1288 (1989); Zimmer et al., Nature, 338:150 (1989); whichare incorporated herein by reference). Homologous recombinationtechniques are well known in the art. Homologous recombination replacesthe native (endogenous) gene with a recombinant or mutated gene toproduce an animal that cannot express native (endogenous) protein butcan express, for example, a mutated protein which results in alteredexpression of DR5 or TRAIL-R3 proteins.

[0099] In contrast to homologous recombination, microinjection addsgenes to the host genome, without removing host genes. Microinjectioncan produce a transgenic animal that is capable of expressing bothendogenous and exagenous DR5 or TRAIL-R3 protein. Inducible promoterscan be linked to the coding region of nucleic acids to provide a meansto regulate expression of the transgene. Tissue specific regulatoryelements can be linked to the coding region to permit tissue-specificexpression of the transgene. Transgenic animal model systems are usefulfor in vivo screening of compounds for identification of specificagents, i.e., agonists and antagonists, which activate or inhibitprotein responses.

[0100] Invention nucleic acids, oligonucleotides (including antisense),vectors containing same, transformed host cells, polypeptides andcombinations thereof, as well as antibodies of the present invention,can be used to screen compounds in vitro to determine whether a compoundfunctions as a potential agonist or antagonist to invention DR5 orTRAIL-R3 proteins. These in vitro screening assays provide informationregarding the function and activity of invention DR5 or TRAIL-R3proteins, which can lead to the identification and design of compoundsthat are capable of specific interaction with one or more types ofpolypeptides, peptides or proteins.

[0101] Apoptosis plays a significant role in numerous pathologicalconditions in that programmed cell death is either inhibited, resultingin increased cell survival, or enhanced which results in the loss ofcell viability. Examples of pathological conditions resulting fromincreased cell survival include cancers such as lymphomas, carcinomasand hormone dependent tumors. Such hormone dependent tumors include, forexample, breast, prostate and ovarian cancer. Autoimmune diseases suchas systemic lupus erythematosus and immune-mediated glomerulonephritisas well as viral infections such as herpes virus, poxvirus andadenovirus also result from increased cell survival or the inhibition ofapoptosis.

[0102] In contrast, apoptotic diseases where enhanced programmed celldeath is a prevalent cause generally includes, for example, degenerativedisorders such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Retinitis pigmentosa, and Cerebellar degeneration.Other diseases associated with increased apoptosis include, for example,myelodysplastic syndromes such as aplastic anemia and ischemic injuryincluding myocardial infarction, stroke and reperfusion injury.

[0103] The DR5 or TRAIL-R3 encoding nucleic acids and polypeptides ofthe invention can be used to diagnose, treat or reduce the severity ofcell death mediated diseases such as those described above as well asother diseases mediated by either increased or decreased programmed celldeath. Additionally, the DR5 or TRAIL-R3 encoding nucleic acids andpolypeptides of the invention can be used to screen for pharmaceuticalcompounds and macromolecules which inhibit or promote DR5 or TRAIL-R3mediated apoptosis.

[0104] For example, the DR5 or TRAIL-R3 encoding nucleic acids,polypeptides and functional fragments thereof can be used to diagnose,or to generate reagents to diagnose diseases mediated or characterizedby programmed cell death. Diagnosis can be by nucleic acid probehybridization with DR5 or TRAIL-R3 containing nucleotide sequences,antibody or ligand mediated detection with DR5 or TRAIL-R3 bindingagents or by enzyme catalysis of detectable DR5 or TRAIL-R3 substrates.Such methods are routine to those skilled in the art. Detection can beperformed ex vivo, for example, by removing a cell or tissue sample froman individual exhibiting or suspected of exhibiting a cell deathmediated disease. Correlation of increased DR5 or TRAIL-R3 expression oractivity may be indicative of diseases characterized by enhancedprogrammed cell death whereas correlation of decreased DR5 or TRAIL-R3expression or activity may be indicative of diseases characterized bythe inhibition of programmed cell death.

[0105] Thus, in accordance with still another embodiment of the presentinvention, there is provided a method for identifying compounds that maybind to DR5 or TRAIL-R3 proteins such as, for example, antibodies,binding agents, and the like. For example, the invention DR5 or TRAIL-R3proteins may be employed in a competitive binding assay. Such an assaycan accommodate the rapid screening of a large number of compounds todetermine which compounds, if any, are capable of binding to DR5 orTRAIL-R3 proteins. Subsequently, more detailed assays can be carried outwith those compounds found to bind, to further determine whether suchcompounds act as modulators, e.g., agonists or antagonists, of inventionproteins.

[0106] Thus, in another embodiment of the invention, there is provided abioassay for identifying compounds that modulate the activity ofinvention DR5 or TRAIL-R3 proteins. According to this method, inventionDR5 or TRAIL-R3 proteins, preferably membrane bound, may be contactedwith TRAIL ligand in the presence and in the absence of a test-compound;the activity of the DR5 or TRAIL-R3 proteins is monitored subsequent tothe contact with the test compound, and those test-compounds that maycause either the increase or decrease of apoptosis in cellular systemshaving membrane bound DR5 or TRAIL-R3 proteins therein may be identifiedas functional agents for modulating DRS or TRAIL-R3 proteins.

[0107] In accordance with another embodiment of the present invention,transformed host cells (either completely-intact or semi-intact cells)that recombinantly express invention DR5 or TRAIL- R3 proteins can becontacted with a test compound, and the modulating effect(s) thereof canthen be evaluated by comparing the relative levels of apoptosismodulation in the presence and absence of test compound, or by comparingthe response of test cells or control cells (i.e., cells that do notexpress DR5 or TRAIL-R3 proteins), to the presence of the compound.

[0108] As used herein, a compound or a signal that “modulates theactivity” of invention DR5 or TRAIL-R3 proteins refers to a compound ora signal that may alter the activity of DRS or TRAIL-R3 proteins so thatthe activity of the invention DR5 or TRAIL-R3 proteins is different inthe presence of the compound or signal than in the absence of thecompound or signal. In particular, such compounds or signals includeagonists and antagonists. An agonist encompasses a compound or a signalthat may activate or increase the function of invention DR5 or TRAIL-R3proteins. Alternatively, an antagonist includes a compound or signalthat may interfere with, inhibit or otherwise decrease DR5 or TRAIL-R3protein function. Typically, the effect of an antagonist may be observedas a blocking of agonist-induced protein activation. Antagonists includecompetitive and non-competitive antagonists. A competitive antagonist(or competitive blocker) interacts with or near the site specific forTRAIL binding. A non-competitive antagonist or blocker inactivates thefunction of the polypeptide by interacting with a site other than theapoptosis modulating region of invention DR5 or TRAIL-R3 proteins.

[0109] As understood by those of skill in the art, assay methods foridentifying compounds that modulate DR5 or TRAIL-R3 activity generallyrequire comparison to a control. One type of a “control” is a cell orculture that is treated substantially the same as the test cell or testculture exposed to the compound, with the distinction that the “control”cell or culture is not exposed to the compound. For example, a type of“control” cell or culture may be a cell or culture that is identical tothe transfected cells, with the exception that the “control” cell orculture do not express native proteins. Accordingly, the response of thetransfected cell to compound may be compared to the response (or lackthereof) of the “control” cell or culture to the same compound under thesame reaction conditions.

[0110] In another embodiment of the present invention, there is provideda bioassay for evaluating whether test compounds are capable of actingas agonists or antagonists for DR5 or TRAIL-R3 proteins, wherein saidbioassay comprises:

[0111] (a) culturing cells containing: DNA which expresses a TRAILreceptor selected from DR5 or TRAILR3, or functional modified formsthereof, wherein said culturing is carried out in the presence of atleast one compound whose ability to modulate apoptotic activity of TRAILreceptors is sought to be determined, and thereafter

[0112] (b) monitoring said cells for either an increase or decrease inthe level of apoptosis.

[0113] Such an assay can be carried out in the presence or absence ofTRAIL ligand. Methods are well-known in the art for measuring apoptosiscan be employed in bioassays described herein to identify agonists andantagonists of DR5 or TRAIL-R3 proteins. For example, the methodsdescribed in Example IV can be used to evaluate the apoptotic activityof recombinant DR5 or TRAIL-R3 proteins or mutants and/or analogsthereof, expressed in mammalian host cells.

[0114] In addition, the occurrence of apoptosis in cell-free systems canbe assessed by detecting the relative levels of: caspase processing(i.e., the cleavage of the pro-caspase to active forms; see, e.g.,Casciola-Rosen et al., 1996, J. Exp. Med.,183:1957-1964; Tewari et al,1995, J. Biol. Chem., 32:18738-18741; Tewari et al, 1995, Cell,81:801-809), caspase activation, cytosolic substrate cleavage, therelease of cytochrome-c from mitochondria, and the like. Exemplarycytosolic substrates that are cleaved as a result of apoptosis are setforth in Table 1, and include: fodrin, CPP32, PKC6, and the like.

[0115] When nuclei are present in the bioassays described herein, theoccurrence of apoptosis can be assessed by, in addition to the methodsdescribed above, detecting: chromatin condensation, shrinkage andfragmentation of the nuclei, and the like (see, for example, Zanzami etal., J. Exp. Med., 183:1533-1544 (1995); Newmeyer et al., Cell79:353-364 (1994)). In addition, nuclear substrates that are cleaved asa result of apoptosis are also set forth in Table 1, and include: DNAtopoisomerase (Liu, Ann. Rev. Biochem. 58, 351-375 1989), lamin B(Lazebnik et al., Proc. Natl. Acad. Sci. USA 92, 9042-9046. 1995), NuMA(Compton, 1994), PARP (Lazebnik et al., Supra, 1994), and U1-70kDa(Casciola-Rosen et al., J. Biol. Chem. 49, 30757-30760. 1994), and thelike.

[0116] As used herein, “ability to modulate apoptotic activity of TRAILreceptors” refers to a compound that has the ability to either induce(agonist) or inhibit (antagonist) apoptosis mediated by DR5 or TRAIL-R3proteins.

[0117] In another embodiment of the present invention, the bioassay forevaluating whether test compounds are capable of acting as antagonistsfor DR5 and TRAIL-R3 proteins of the invention, or functional modifiedforms of said DR5 and TRAIL-R3 proteins, comprises:

[0118] (a) culturing cells containing: DNA which expresses DR5 andTRAIL-R3 proteins, or functional modified forms thereof, wherein saidculturing is carried out in the presence of: increasing concentrationsof at least one compound whose ability to inhibit apoptotic activity ofDR5 and TRAIL-R3 proteins is sought to be determined, and a fixedconcentration of TPAIL; and thereafter

[0119] (b) monitoring in said cells the level of apoptosis as a functionof the concentration of said compound, thereby indicating the ability ofsaid compound to inhibit DR5 or TRAIL-R3 mediated apoptotic activity.

[0120] In step (a) of the above-described antagonist bioassay, culturingmay also be carried out in the presence of:

[0121] fixed concentrations of at least one compound whose ability toinhibit apoptotic activity of DR5 and TRAIL-R3 proteins is sought to bedetermined, and

[0122] an increasing concentration of TRAIL.

[0123] Host cells contemplated for use in the bioassay(s) of the presentinvention include MCH6 cells, 293 cells, CV-1 cells, COS cells, HeLacells, and the like. Presently, preferred host cells for carryinginvention bioassays are HeLa cells as described in Example VI.

[0124] Also contemplated in yet another embodiment of the presentinvention, is a method for modulating the apoptotic activity mediated byDR5 or TRAIL-R3 proteins, said method comprising:

[0125] contacting an DR5 or TRAIL-R3 protein with an effective,modulating amount of an agonist or antagonist identified by theabove-described bioassays.

[0126] The present invention also contemplates therapeutic compositionsuseful for practicing the therapeutic methods described herein. Forexample, the above described DR5 or TRAIL-R3 polypeptides can also beformulated into pharmaceutical compositions known within the art for thetreatment of programmed cell death mediated diseases characterized byincreased or decreased cell survival and proliferation. Functionalfragments and peptides such as the extracellular TRAIL-binding domainsand the cytoplasmic death domain of DR5 or TRAIL-R3 can similarly beformulated for the treatment of such diseases associated with increasedor decreased cell survival and proliferation. Additionally, moleculesthat interact with DR5 or TRAIL-R3 can additionally be used to modulateDR5 or TRAIL-R3 mediated apoptosis. Administration of DR5 polypeptidesand functional fragments thereof may induce or inhibit apoptosis intreated cells, and may eliminate those cells characterized by increasedcell survival or proliferation.

[0127] Treatment or reduction of the severity of cell death mediateddiseases can also be accomplished by introducing expressible nucleicacids encoding DRS or TRAIL-R3 polypeptides or functional fragmentsthereof into cells characterized by such diseases. Elevated syntheticrates of DRS or TRAIL-R3 may be achieved, for example, by usingrecombinant expression vectors and gene transfer technology. Similarly,treatment or reduction of the severity of cell death mediated diseasesmay also be accomplished by introducing and expressing antisense DR5 orTRAIL-R3 nucleic acids so as to inhibit the synthesis rates of DR5 orTRAIL-R3. Such methods are well known within the art and are describedherein with reference to recombinant viral vectors. Other vectorscompatible with the appropriate targeted cell can accomplish the samegoal and therefore can be substituted in the methods described herein inplace of recombinant viral vectors.

[0128] Therapeutic compositions of the present invention contain aphysiologically compatible carrier together with DR5 or TRAIL-R3polypeptides or functional fragments thereof, a DR5 or TRAIL-R3modulating agent, or an anti-DR5 or anti-TRAIL-R3 antibody, as describedherein, dissolved or dispersed therein as an active ingredient.

[0129] As used herein, the terms “pharmaceutically acceptable”,“physiologically acceptable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset, and thelike.

[0130] The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well known in theart. Typically such compositions are prepared as injectables either asliquid solutions or suspensions; however, solid forms suitable forsolution, or suspension, in liquid prior to use can also be prepared.The preparation can also be emulsified.

[0131] The active ingredient can be mixed with excipients that arepharmaceutically acceptable and compatible with the active ingredient inamounts suitable for use in the therapeutic methods described herein.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like, as well as combinations of any two or morethereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and the like, which enhance the effectiveness ofthe active ingredient.

[0132] The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable nontoxic salts include the acid additionsalts (formed with the free amino groups of the polypeptide) that areformed with inorganic acids such as, for example, hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolicacid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid,naphthalene sulfonic acid, sulfanilic acid, and the like.

[0133] Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and the like; and organic bases such asmono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine,diisopropyl amine, methyl amine, dimethyl amine, and the like) andoptionally substituted ethanolamines (e.g., ethanolamine,diethanolamine, and the like).

[0134] Physiologically tolerable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

[0135] Liquid compositions can also contain liquid phases in addition toand to the exclusion of water. Exemplary additional liquid phasesinclude glycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

[0136] As described herein, an “effective amount” is a predeterminedamount calculated to achieve the desired therapeutic effect, e.g., tomodulate the TRAIL mediated apoptotic activity of an invention DR5 orTRAIL-R3 protein. The required dosage will vary with the particulartreatment and with the duration of desired treatment. Such dosages areknown or can be easily determined by those skilled in the art.Administration may be accomplished, for example, by intravenous,interperitonal or subcutaneous injection. Administration may beperformed in a variety of different regimes, which include single highdose administration or repeated small dose administration, or acombination of both. The dosing may depend on the cell type, progressionof the disease and overall health of the individual, and will be knownor can be determined by those skilled in the art.

[0137] It is contemplated that dosages between about 10 micrograms andabout 1 milligram per kilogram of body weight per day may be used fortherapeutic treatment. It may be particularly advantageous to administersuch compounds in depot or long-lasting form as discussed herein. Atherapeutically effective amount is typically an amount of an DR5- orTRAIL-R3-modulating agent or compound identified herein that, whenadministered in a physiologically acceptable composition, is sufficientto achieve a plasma concentration of from about 0.1 μg/ml to about 100μg/ml, preferably from about 1.0 μg/ml to about 50 μg/ml, morepreferably at least about 2 μg/ml and usually 5 to 10 μg/ml.

[0138] All U.S. patents and all publications mentioned herein areincorporated in their entirety by reference thereto. The invention willnow be described in greater detail by reference to the followingnon-limiting examples.

EXAMPLES

[0139] Unless otherwise stated, the present invention was performedusing standard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methodsin Molecular Biology, Elsevier Science Publishing, Inc., New York, USA(1986); or Methods in Enzymology: Guide to Molecular Cloning TechniquesVol 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., SanDiego, USA (1987).,

Example I Cloning and Characterization of DR5 and TRAIL-R3

[0140] This example demonstrates the identification and cloning of DR5and TRAIL-R3 cDNA. Briefly, several EST clones were identified and their3′ and 5′ sequences were compiled. Based on the compiled sequences, PCRprimers were generated and used to clone two cDNAs which encode twonovel members of the TRAIL-receptor family (FIGS. 1A and 1B).

[0141] To identify novel members of the TNF-receptor family, an approachcombining information from the GenBank database of human expressedsequence tags (ESTs) and PCR was employed (see Alnemri E. S. et al.(1996) Proc. Natl. Sci. USA. 93, 7464-7469; Femandez-Alnerni et al.,Cancer Res. 5S:2737-2742 (1995); and Fernandez-Alnemri et al., CancerRes. 55:6045-6052 (1995)). Initially, a search of the GenBank EST database for sequences related to TRAIL receptor-1, also referred to as“DR4”, identified several EST clones, which were used to derive 5′ and3′ PCR primers to clone related cDNAs.

[0142] The EST sequences used to clone DR5 were identified as humanGenBank EST clone 650744 (Accession Nos. AA223122 and AA223238) andclone 664665 (Accession Nos. AA232424 and AA232440). These EST cloneswere used to design primers at the extreme 5′ and 3′ ends of the DR5coding region. The 5′ primer employed to amplify DR5 cDNA correspondedto the oligonucleotide set forth as nucleotides 1-18 of SEQ ID NO:1. The3′ primer used corresponds to the oligonucleotide complementary tonucleotides 1219-1236 of SEQ ID NO: 1.

[0143] Similarly, the EST sequences used to clone TRAIL-R3 wereidentified as human GenBank EST clone 470799 (Accession No. AA031883),clone 129137 (Accession Nos. R10995 and R10996) and clone 504745(Accession Nos. AA150849 and AA150541). These EST clones were used todesign primers at the extreme 5′ and 3′ ends of the TRAIL-R3 codingregion. The 5′ primer employed to amplify TRAIL-R3 cDNA co-responded tothe oligonucleotide set forth as nucleotides 1-18 of SEQ ID NO:3. The 3′primer used corresponds to the oligonucleotide complementary tonucleotides 883-900 SEQ ID NO:3.NO:1.

[0144] A 10 μl aliquot of human Jurkat λ Uni-Zap™ XR cDNA library(Femandez-Alnemri et al., J. Biol. Chem. 269:30761-30764 (1994))containing approximately 10⁸ pfu was denatured at 99° C. for 5 min. andused as a template for PCR amplification with the above-described primerpairs for cloning DR5 and TRAIL-R3 cDNA. The full-length amplificationproducts were cloned into a small-cut pBluescript II KS⁺ vector andsequenced.

[0145] To confirm the sequence of the PCR-amplified DR5 and TRAIL-R3cDNA, the cloned cDNA was then excised from the vector, radiolabeled andused to screen the original Jurkat X Uni-Zap™ XR cDNA library for fulllength cDNA clones. Positive A plones were purified, rescued into thepBluescript II SK plasmid vector and sequenced for confirmation.

[0146] FIGS. 1A-1D show the sequence analysis and tissue distribution ofinvention DR5 and TRAIL-R3 proteins. The predicted amino acid sequenceof human DR5 and TRAIL-R3 is set forth in FIGS. 1A and 1B, respectively.The mature DR5 and TRAIL-R3 proteins start at Glu+1 and Tyr+1 (indicatedby black diamonds), respectively. The signal peptide and transmembranedomains are single- and double-underlined, respectively. The fiveidentical repeats in the extracellular domain of TRAIL-R3 (FIG. 1B) aremarked by black triangles. The intracellular cytoplasmic death domain ofDR5 (FIG. 1A) is boxed.

[0147] The DR5 cDNA encodes a protein of 411 amino acids (SEQ ID NO:2)with an overall −59% amino acid identity to DR4 (FIG. 1A). Its domainstructure is highly related to DR4 and the other members of theTNF-receptor family. DR5 contains a putative N-terminal signal peptide(amino acids −51 to −1 of FIG. 1A) followed by an extracellular domaincontaining two cysteine-rich pseudorepeats. Following the extracellulardomain is a transmembrane domain (amino acids 132-152 of FIG. 1A) and acytoplasmic domain. Within the cytoplasmic domain there is a stretch of67-amino acids (amino acids 273-339 of FIG. 1A) comprising a deathdomain homology region (FIG. 1C). Based on these criteria and itsapoptotic activity (see Example IV below) this TRAIL-receptor proteinwas designated death receptor-5 (DR5) (also referred to herein asTRAIL-R2).

[0148] The TRAIL-R3 cDNA encodes a protein of 299 amino acids with anoverall ˜40% and 36% amino acid identity to DR4 and DR5, respectively(FIG. 1B). TRAIL-R3 contains an N-terminal signal peptide (amino acids−63 to −1 of FIG. 1B) followed by an extracellular domain containing twocysteine-rich pseudorepeats and five nearly identical PAAEETMN(T)TSPGTPArepeats (amino acids 139-153, 154-168, 169-183, 184-198, and 199-213 ofFIG. 1B). Following the extracellular domain is a C-terminaltransmembrane domain (amino acids 217-236 of FIG. 1B). Unlike DR4 andDR5, TRAIL-R3 does not contain a cytoplasmic domain. Based on thesecriteria and its ability to bind TRAIL (see Example III below), thisprotein was designated TRAIL-R3.

[0149]FIG. 11C shows a colinear alignment of the death domains ofmembers of the TNF receptor family. Identical residues in at least threeout of six sequences are shaded. The death domain of DR5 is 64, 30, 30,20, 31% identical to the corresponding domains in DR4, DR3, TNFR-1, Fasand CAR1, respectively.

Example II expression analyst. of DR5 and TRAIL-R3 mRNA

[0150] This example demonstrates the mRNA expression patterns of DR5 andTRAIL-R3 in normal and tumor cells.

[0151]FIG. 11D shows a Northern blot analysis of the expression of DR5(upper panels) and TRAIL-R3 (lower panels) mRNAs in normal tissues andtumor cell lines. X-ray film exposure time in the two lower panels andthe upper left panel was for 48 hours, whereas in the upper right panelwas for 2 hours. The cell lines assayed were: HL-60, promyelocyticleukemia; HeLa cell S3, K-562, chronic myelogenous leukemia; MOLT-4,lymphoblastic leukemia; Raji, Burkitt's lymphoma; SW480, colorectaladenocarcinoma; A549, lung carcinoma; G361, melanoma. Numbers on theright in FIG. 11D indicate kilobases.

[0152] Northern blot analysis of equivalent amounts of mRNA samples fromnormal human tissues and tumor cell lines, with a DR5 riboprobe,detected a ˜4 kb transcript in all the samples (FIG. 1D, upper panels).Surprisingly, the amount of DR5 transcript was at least a 100-fold morein most tumor cell lines than in normal tissues. Autoradiography forless than 2 h was sufficient to detect the DR5 message in tumor celllines, compared to 48 h in the case of the normal tissues. Otherabnormal tissues such testes, ovary, colon, small intestine and lymphoidtissues had detectable but low expression of DR5 transcript, similar tothat observed in the normal tissues shown in FIG. 1D.

[0153] The TRAIL-R3 riboprobe detected ˜5 kb message in both normalhuman tissues and tumor cell lines (FIG. 1D, lower panels). Asignificantly elevated expression of TRAIL-R3 mRNA in normal compared totumor cells was observed. Given the activities of these two receptors(see below), this could explain the high sensitivity of tumor cell linesto TRAIL compared to normal cells (Davis T. D. et al. (1995) Immunity 3,673-682; Ashkenazi A. et al. (1996) J. Bio. Chem. 271, 12687-12690;Ashkenazi A. et al. (1996) Curr. Biol. 6, 750-752).

Example III Trail Binding Assay

[0154] This example demonstrates that DR5 and TRAIL-R3 are receptors forthe cytotoxic ligand TRAIL and can block TRAIL-induced apoptosis.

[0155] To generate C-terminal Fc-tagged receptors, PCR generated cDNAsencoding Fas (residues −16-158), DR4 (residues 86-217, with N-terminalFas signal peptide-Flag tag), DR5 (residues −51-133) and TRAIL-R3(residues -63-217) extracellular domains were inserted into a modifiedpcDNA3 vector that allowed for inframe fusion with the Fc portion of themouse IgG.

[0156] Recombinant soluble TRAIL with N-terminal T7 and His6 tags wasobtained by Ni-affinity purification from bacteria transformed with apET28c-TRAIL (residues 95-281) vector. Receptor-Fc chimeras wereobtained by harvesting conditioned media of 293 cells transfected withconstructs encoding Fas-, DR4-, DR5-or TRAIL-R3-Fc fusion proteins asdescribed (Chinnaiyan, A. M. et al. (1997) Science 276, 111-113).Binding of TRAIL to the receptor-Fe chimeras was performed as described(Chinnaiyan, A. M. et al. (1997) Science 276, 111-113).

[0157]FIG. 2 shows that the extracellular domains of DR5 and TRAIL-R3bind TRAIL and can block TRAIL-induced apoptosis. FIG. 2A shows theresults of an assay in which conditioned media from cultures of 293cells transfected for 72 h with empty vector (lane 1), or DR5 (lane 2),DR4 (lane 3), Fas (lane 4) or TRAIL-R3 (TR3-Fc) (lane 5) extracellulardomain-Fe fusion proteins, were incubated with purified solubleT7-His6-TRAIL and then immunoprecipitated with anti-mouse IgG-agarose.After extensive washing the samples were analyzed by SDS-PAGE andimmunoblotted with a horseradish peroxidase (HRP)-conjugated T7-antibody(upper panel). The corresponding receptor-Fc fusions in the conditionedmedia were also immunoblotted with anti-mouse Fc antibody (lower panel).

[0158]FIG. 2B shows the results of an assay in which aliquots ofconditioned media containing receptor-Fc fusion proteins or no fusionprotein (vector) were incubated with equivalent amount of soluble TRAIL(250 ng/ml) and then added to MCF7 cells. Cells were stained 8 hourslater with propidium iodide and the nuclei were examined by fluorescencemicroscopy. The graph shows the percentage of apoptotic nuclei (mean±SD) as a function of total nuclei counted under each condition (n=3).

[0159] As set forth above, the extracellular-ligand binding domains ofFas, DR4, DR5 and TRAIL-R3 were expressed as fusion proteins with the Fcregion of mouse IgG (FIG. 2A, lower panel). As shown in FIG. 2A, upperpanel, DR4-Fc, DR5-Fc and TRAIL-R3-Fc were all capable of binding TRAILto the same extent (lanes 2, 3 and 5). As expected Fas-Fc was unable tobind TRAIL (lane 4). Furthermore, DR4-Fc, DR5-Fc and TRAIL-R3-FC, butnot Fas-Fc, were capable of blocking TRAIL-induced apoptosis in MCF7cells (FIG. 2B). These data indicate that, like DR4, DR5 and TRAIL-R3are receptors for TRAIL.

Example IV Apoptotic Activity of DR5 and TRAIL-R3

[0160] This example demonstrates that DR5 but not TRAIL-R3 inducesapoptosis in human cells.

[0161] For apoptosis assays the mammalian double expression vector pRSC(Akporiaye E. T. et al. (1997) BioTech. 22, 68) was used, which allowsfor expression of lacZ under the RSV promoter and the test cDNA (DR4,DR4A, DR5, DRSA, TRAIL-R3) under the CMV promoter. CrmA, FLAME-1,caspase-8-DN (C345A) or caspase-10-DN (C358A) (Alnemri E. S. et al.(1997) J. Biol. Chem. 272, 18542-18545) were expressed using pcDNA3(Invitrogen).

[0162]FIG. 3 illustrates that expression of DR5 but not TRAIL-R3 inducesapoptosis in human cells. MCF7 (FIG. 3A) and 293 (FIG. 3B) cells weretransfected with the indicated pRSC-lacZ constructs. Thirty hours aftertransfection cells were stained with βgal and examined for morphologicalsigns of apoptosis. The graphs show the percentage of round blueapoptotic cells (mean ±SD) as a function of total blue cells under eachcondition (>23).

[0163]FIG. 3C shows that ectopic expression of TRAIL-R3 attenuatesTRAIL-induced apoptosis in MCF7 cells. MCF7 cells were transfected withTRAIL-R3 or vector alone for 36 h, then treated with soluble TRAIL (250ng/ml) for 8 h. The data are represented as in FIGS. 3A and 3B, aftersubtracting the background killing (12-15%) as a result of transfection.

[0164]FIGS. 3D and 3E show that DR4-induced and DR5-induced apoptosis isinhibited by the caspase inhibitors, z-VAD-fmk and CrmiA (FIG. 3D), andby the dominant negative inhibitors, caspase-8-DN, caspase-10-DN andFLAME-1 (FIG. 3E). MCF7 cells were transfected with DR4 or DR5expression constructs in the presence of z-VAD-fink (20 μM) orco-transfected with a four-fold excess of a CrmA, caspase-8-DN,caspase-10-DN or FLAME-I construct or an empty vector. The data arerepresented as in FIGS. 3A and 3B.

[0165] It is known that ectopic expression of death domain-containingmembers of the TNF-receptor family induces apoptosis in aligand-independent manner. Consistent with this observation, it has beenfound that transient expression of DR5 in MCF7 cells or 293 cellstriggers apoptosis (FIGS. 3A and 3B). The level of apoptosis inductionwas similar to that observed with DR4 (FIG. 3A). DR5 induction ofapoptosis was dependent on the presence of the cytoplasmic death domain,as deletion of this domain abolished the ability of DR4 and DR5 toinduce apoptosis (FIGS. 3A and 3B). In contrast, TRAIL-R3 which does notnaturally contain a death domain was incapable of inducing apoptosis(FIGS. 3A and 3B). Interestingly, transient expression of TRAIL-R3 inMCF7 cells significantly blocked TRAIL-induced apoptosis (FIG. 3C),suggesting that TRAIL-R3 functions as an antagonistic decoy receptor.

[0166] Similar to DR4 and other TNF-receptor family members, DR5-inducedapoptosis was efficiently blocked by the caspase inhibitors z-VAD-fmkand CrmA (FIG. 3D). DR4-induced and DR5-induced apoptosis was alsosignificantly inhibited by the dominant negative inhibitors FLAME-1(also known as Casper, FLIP, I-FLICE, CASH) (Alnemri E. S. et al. (1997)J. Biol. Chem. 272, 18542-18545; Wallach, D. (1997) Nature 388, 123-125;and references therein), caspase-8-DN and caspase-10-DN (FIG. 3E). Amongthese, caspase-10-DN was the most effective in blocking DR4-included andDR5-induced apoptosis. Inhibition of DR4-induced and DR5-inducedapoptosis by FLAME-1 is consistent with recent observations thatTRAIL-induced apoptosis is blocked by expression of FLIP (FLAME-1)(Bodmer J. L. et al. (1997) Nature 388, 190-195). These data alsosuggest that the upstream caspases-8 and -10 are involved in both theDR4 and DR5 death signaling pathways.

Example V In vivo Interactions of DR5

[0167] This example demonstrates that DR4 and DR5 recruit caspase-8,caspase-10 and FLAME-1 to the death signaling pathway.

[0168] T7-epitope tagging was done as described recently (Alnemri E. S.et al. (1997) J. Biol. Chem. 272, 18542-18545. To generate N-terminalFlag-tagged receptor and receptor mutants, PCR generated cDNAs encodingFas, DR4, DR4A (residues 86-351), DR5, DR56 (residues 1-268) wereinserted in a modified pcDNA-3 vector that allowed for inframe fusionwith a Flag-epitope tag that is preceded by Fas-signal peptide.

[0169] Death domain containing adaptor molecules such as FADD/MORTI,CRADD/RAIDD, TRADD and RIP are recruited by some members of theTNF-receptor family to engage the upstream caspases (Nagata, S. (1997)Cell 88, 355-365); Alnenri E. S. et al. (1997) J. Biol. Chem. 272,18542-18545). Using co-immunoprecipitation experiments, DR5 was assayedto determine whether it could interact with these molecules to transmitthe apoptotic signal.

[0170]FIG. 4 illustrates the in vivo interactions of DR5. FIG. 4A showsthat DR5 does not recruit FADD or CRADD. 293 cells were transfected withexpression plasmids encoding T7-epitope tagged CRADD or FADD andFlag-epitope tagged Fas or DR5. After 36 h, extracts were prepared andimmunoprecipitated with a monoclonal antibody to the Flag-epitope. Theimmunoprecipitates (upper panel) and the corresponding cellular extracts(lower panel) were analyzed by SDS-PAGE and immunoblotted with aHRP-conjugated T7-antibody.

[0171]FIG. 4B shows that caspase-8, caspase-10 and FLAME-1 are recruitedto the Fas, DR4 and DR5 signaling complexes. Briefly, 293 cells wereco-transfected with the indicated Flag constructs and plasmids encodingT7-caspase-10 (upper panel), T7-caspase-8 (middle panel) or T7-FLAME-1(lower panel) and then immunoprecipitated and detected as in FIG. 4A.

[0172] Unlike Fas, DR5 did not interact with FADD or CRADD (FIG. 4A),nor with RIP or TRADD. A similar observation was reported with DR4 (Panet al., 1997, Science 276, 111-113). Interestingly, full length Fas, DR4and DR5, but not death domain-deleted mutants, were all capable offorming complexes with caspase-8, caspase-10 and FLAME-I (FIG. 4B).Since these proteins do not interact directly, it is believed thatformation of these complexes would require an adaptor molecule distinctfrom FADD.

[0173] Although the invention has been described with reference to theexamples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the claims.

1 6 1 1236 DNA Homo sapiens CDS (1)..(1233) 1 atg gaa caa cgg gga cagaac gcc ccg gcc gct tcg ggg gcc cgg aaa 48 Met Glu Gln Arg Gly Gln AsnAla Pro Ala Ala Ser Gly Ala Arg Lys 1 5 10 15 agg cac ggc cca gga cccagg gag gcg cgg gga gcc agg cct ggg ctc 96 Arg His Gly Pro Gly Pro ArgGlu Ala Arg Gly Ala Arg Pro Gly Leu 20 25 30 cgg gtc ccc aag acc ctt gtgctc gtt gtc gcc gcg gtc ctg ctg ttg 144 Arg Val Pro Lys Thr Leu Val LeuVal Val Ala Ala Val Leu Leu Leu 35 40 45 gtc tca gct gag tct gct ctg atcacc caa caa gac cta gct ccc cag 192 Val Ser Ala Glu Ser Ala Leu Ile ThrGln Gln Asp Leu Ala Pro Gln 50 55 60 cag aga gtg gcc cca caa caa aag aggtcc agc ccc tca gag gga ttg 240 Gln Arg Val Ala Pro Gln Gln Lys Arg SerSer Pro Ser Glu Gly Leu 65 70 75 80 tgt cca cct gga cac cat atc tca gaagac ggt aga gat tgc atc tcc 288 Cys Pro Pro Gly His His Ile Ser Glu AspGly Arg Asp Cys Ile Ser 85 90 95 tgc aaa tat gga cag gac tat agc act cactgg aat gac ctc ctt ttc 336 Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His TrpAsn Asp Leu Leu Phe 100 105 110 tgc ttg cgc tgc acc agg tgt gat tca ggtgaa gtg gag cta agt ccc 384 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly GluVal Glu Leu Ser Pro 115 120 125 tgc acc acg acc aga aac aca gtg tgt cagtgc gaa gaa ggc acc ttc 432 Cys Thr Thr Thr Arg Asn Thr Val Cys Gln CysGlu Glu Gly Thr Phe 130 135 140 cgg gaa gaa gat tct cct gag atg tgc cggaag tgc cgc aca ggg tgt 480 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg LysCys Arg Thr Gly Cys 145 150 155 160 ccc aga ggg atg gtc aag gtc ggt gattgt aca ccc tgg agt gac atc 528 Pro Arg Gly Met Val Lys Val Gly Asp CysThr Pro Trp Ser Asp Ile 165 170 175 gaa tgt gtc cac aaa gaa tca ggc atcatc ata gga gtc aca gtt gca 576 Glu Cys Val His Lys Glu Ser Gly Ile IleIle Gly Val Thr Val Ala 180 185 190 gcc gta gtc ttg att gtg gct gtg tttgtt tgc aag tct tta ctg tgg 624 Ala Val Val Leu Ile Val Ala Val Phe ValCys Lys Ser Leu Leu Trp 195 200 205 aag aaa gtc ctt cct tac ctg aaa ggcatc tgc tca ggt ggt ggt ggg 672 Lys Lys Val Leu Pro Tyr Leu Lys Gly IleCys Ser Gly Gly Gly Gly 210 215 220 gac cct gag cgt gtg gac aga agc tcacaa cga cct ggg gct gag gac 720 Asp Pro Glu Arg Val Asp Arg Ser Ser GlnArg Pro Gly Ala Glu Asp 225 230 235 240 aat gtc ctc aat gag atc gtg agtatc ttg cag ccc acc cag gtc cct 768 Asn Val Leu Asn Glu Ile Val Ser IleLeu Gln Pro Thr Gln Val Pro 245 250 255 gag cag gaa atg gaa gtc cag gagcca gca gag cca aca ggt gtc aac 816 Glu Gln Glu Met Glu Val Gln Glu ProAla Glu Pro Thr Gly Val Asn 260 265 270 atg ttg tcc ccc ggg gag tca gagcat ctg ctg gaa ccg gca gaa gct 864 Met Leu Ser Pro Gly Glu Ser Glu HisLeu Leu Glu Pro Ala Glu Ala 275 280 285 gaa agg tct cag agg agg agg ctgctg gtt cca gca aat gaa ggt gat 912 Glu Arg Ser Gln Arg Arg Arg Leu LeuVal Pro Ala Asn Glu Gly Asp 290 295 300 ccc act gag act ctg aga cag tgcttc gat gac ttt gca gac ttg gtg 960 Pro Thr Glu Thr Leu Arg Gln Cys PheAsp Asp Phe Ala Asp Leu Val 305 310 315 320 ccc ttt gac tcc tgg gag ccgctc atg agg aag ttg ggc ctc atg gac 1008 Pro Phe Asp Ser Trp Glu Pro LeuMet Arg Lys Leu Gly Leu Met Asp 325 330 335 aat gag ata aag gtg gct aaagct gag gca gcg ggc cac agg gac acc 1056 Asn Glu Ile Lys Val Ala Lys AlaGlu Ala Ala Gly His Arg Asp Thr 340 345 350 ttg tac acg atg ctg ata aagtgg gtc aac aaa acc ggg cga gat gcc 1104 Leu Tyr Thr Met Leu Ile Lys TrpVal Asn Lys Thr Gly Arg Asp Ala 355 360 365 tct gtc cac acc ctg ctg gatgcc ttg gag acg ctg gga gag aga ctt 1152 Ser Val His Thr Leu Leu Asp AlaLeu Glu Thr Leu Gly Glu Arg Leu 370 375 380 gcc aag cag aag att gag gaccac ttg ttg agc tct gga aag ttc atg 1200 Ala Lys Gln Lys Ile Glu Asp HisLeu Leu Ser Ser Gly Lys Phe Met 385 390 395 400 tat cta gaa ggt aat gcagac tct gcc atg tcc taa 1236 Tyr Leu Glu Gly Asn Ala Asp Ser Ala Met Ser405 410 2 411 PRT Homo sapiens 2 Met Glu Gln Arg Gly Gln Asn Ala Pro AlaAla Ser Gly Ala Arg Lys 1 5 10 15 Arg His Gly Pro Gly Pro Arg Glu AlaArg Gly Ala Arg Pro Gly Leu 20 25 30 Arg Val Pro Lys Thr Leu Val Leu ValVal Ala Ala Val Leu Leu Leu 35 40 45 Val Ser Ala Glu Ser Ala Leu Ile ThrGln Gln Asp Leu Ala Pro Gln 50 55 60 Gln Arg Val Ala Pro Gln Gln Lys ArgSer Ser Pro Ser Glu Gly Leu 65 70 75 80 Cys Pro Pro Gly His His Ile SerGlu Asp Gly Arg Asp Cys Ile Ser 85 90 95 Cys Lys Tyr Gly Gln Asp Tyr SerThr His Trp Asn Asp Leu Leu Phe 100 105 110 Cys Leu Arg Cys Thr Arg CysAsp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125 Cys Thr Thr Thr Arg AsnThr Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 Arg Glu Glu Asp SerPro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 160 Pro Arg GlyMet Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175 Glu CysVal His Lys Glu Ser Gly Ile Ile Ile Gly Val Thr Val Ala 180 185 190 AlaVal Val Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp 195 200 205Lys Lys Val Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly 210 215220 Asp Pro Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp 225230 235 240 Asn Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln ValPro 245 250 255 Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr GlyVal Asn 260 265 270 Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu ProAla Glu Ala 275 280 285 Glu Arg Ser Gln Arg Arg Arg Leu Leu Val Pro AlaAsn Glu Gly Asp 290 295 300 Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp AspPhe Ala Asp Leu Val 305 310 315 320 Pro Phe Asp Ser Trp Glu Pro Leu MetArg Lys Leu Gly Leu Met Asp 325 330 335 Asn Glu Ile Lys Val Ala Lys AlaGlu Ala Ala Gly His Arg Asp Thr 340 345 350 Leu Tyr Thr Met Leu Ile LysTrp Val Asn Lys Thr Gly Arg Asp Ala 355 360 365 Ser Val His Thr Leu LeuAsp Ala Leu Glu Thr Leu Gly Glu Arg Leu 370 375 380 Ala Lys Gln Lys IleGlu Asp His Leu Leu Ser Ser Gly Lys Phe Met 385 390 395 400 Tyr Leu GluGly Asn Ala Asp Ser Ala Met Ser 405 410 3 900 DNA Homo sapiens CDS(1)..(897) 3 atg caa ggg gtg aag gag cgc ttc cta ccg tta ggg aac tct ggggac 48 Met Gln Gly Val Lys Glu Arg Phe Leu Pro Leu Gly Asn Ser Gly Asp 15 10 15 aga gcg ccc cgg ccg cct gat ggc cga ggc agg gtg cga ccc agg acc96 Arg Ala Pro Arg Pro Pro Asp Gly Arg Gly Arg Val Arg Pro Arg Thr 20 2530 caa gac ggc gtc ggg aac cat acc atg gcc cgg atc ccc aag acc cta 144Gln Asp Gly Val Gly Asn His Thr Met Ala Arg Ile Pro Lys Thr Leu 35 40 45aag ttc gtc gtc gtc atc gtc gcg gtc ctg ctg cca gtc cta gct tac 192 LysPhe Val Val Val Ile Val Ala Val Leu Leu Pro Val Leu Ala Tyr 50 55 60 tctgcc acc act gcc cgg cag gag gaa gtt ccc cag cag aca gtg gcc 240 Ser AlaThr Thr Ala Arg Gln Glu Glu Val Pro Gln Gln Thr Val Ala 65 70 75 80 ccacag caa cag agg cac agc ttc aag ggg gag gag tgt cca gca gga 288 Pro GlnGln Gln Arg His Ser Phe Lys Gly Glu Glu Cys Pro Ala Gly 85 90 95 tct cataga tca gaa cat act gga gcc tgt aac ccg tgc aca gag ggt 336 Ser His ArgSer Glu His Thr Gly Ala Cys Asn Pro Cys Thr Glu Gly 100 105 110 gtg gattac acc aac gct tcc aac aat gaa cct tct tgc ttc cca tgt 384 Val Asp TyrThr Asn Ala Ser Asn Asn Glu Pro Ser Cys Phe Pro Cys 115 120 125 aca gtttgt aaa tca gat caa aaa cat aaa agt tcc tgc acc atg acc 432 Thr Val CysLys Ser Asp Gln Lys His Lys Ser Ser Cys Thr Met Thr 130 135 140 aga gacaca gtg tgt cag tgt aaa gaa ggc acc ttc cgg aat gaa aac 480 Arg Asp ThrVal Cys Gln Cys Lys Glu Gly Thr Phe Arg Asn Glu Asn 145 150 155 160 tcccca gag atg tgc cgg aag tgt agc agg tgc cct agt ggg gaa gtc 528 Ser ProGlu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly Glu Val 165 170 175 caagtc agt aat tgt acg tcc tgg gat gat atc cag tgt gtt gaa gaa 576 Gln ValSer Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys Val Glu Glu 180 185 190 tttggt gcc aat gcc act gtg gaa acc cca gct gct gaa gag aca atg 624 Phe GlyAla Asn Ala Thr Val Glu Thr Pro Ala Ala Glu Glu Thr Met 195 200 205 aacacc agc ccg ggg act cct gcc cca gct gct gaa gag aca atg aac 672 Asn ThrSer Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Asn 210 215 220 accagc cca ggg act cct gcc cca gct gct gaa gag aca atg acc acc 720 Thr SerPro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr 225 230 235 240agc ccg ggg act cct gcc cca gct gct gaa gag aca atg acc acc agc 768 SerPro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser 245 250 255ccg ggg act cct gcc cca gct gct gaa gag aca atg acc acc agc ccg 816 ProGly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser Pro 260 265 270ggg act cct gcc tct tct cat tac ctc tca tgc acc atc gta ggg atc 864 GlyThr Pro Ala Ser Ser His Tyr Leu Ser Cys Thr Ile Val Gly Ile 275 280 285ata gtt cta att gtg ctt ctg att gtg ttt gtt tga 900 Ile Val Leu Ile ValLeu Leu Ile Val Phe Val 290 295 4 299 PRT Homo sapiens 4 Met Gln Gly ValLys Glu Arg Phe Leu Pro Leu Gly Asn Ser Gly Asp 1 5 10 15 Arg Ala ProArg Pro Pro Asp Gly Arg Gly Arg Val Arg Pro Arg Thr 20 25 30 Gln Asp GlyVal Gly Asn His Thr Met Ala Arg Ile Pro Lys Thr Leu 35 40 45 Lys Phe ValVal Val Ile Val Ala Val Leu Leu Pro Val Leu Ala Tyr 50 55 60 Ser Ala ThrThr Ala Arg Gln Glu Glu Val Pro Gln Gln Thr Val Ala 65 70 75 80 Pro GlnGln Gln Arg His Ser Phe Lys Gly Glu Glu Cys Pro Ala Gly 85 90 95 Ser HisArg Ser Glu His Thr Gly Ala Cys Asn Pro Cys Thr Glu Gly 100 105 110 ValAsp Tyr Thr Asn Ala Ser Asn Asn Glu Pro Ser Cys Phe Pro Cys 115 120 125Thr Val Cys Lys Ser Asp Gln Lys His Lys Ser Ser Cys Thr Met Thr 130 135140 Arg Asp Thr Val Cys Gln Cys Lys Glu Gly Thr Phe Arg Asn Glu Asn 145150 155 160 Ser Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly GluVal 165 170 175 Gln Val Ser Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys ValGlu Glu 180 185 190 Phe Gly Ala Asn Ala Thr Val Glu Thr Pro Ala Ala GluGlu Thr Met 195 200 205 Asn Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala GluGlu Thr Met Asn 210 215 220 Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala GluGlu Thr Met Thr Thr 225 230 235 240 Ser Pro Gly Thr Pro Ala Pro Ala AlaGlu Glu Thr Met Thr Thr Ser 245 250 255 Pro Gly Thr Pro Ala Pro Ala AlaGlu Glu Thr Met Thr Thr Ser Pro 260 265 270 Gly Thr Pro Ala Ser Ser HisTyr Leu Ser Cys Thr Ile Val Gly Ile 275 280 285 Ile Val Leu Ile Val LeuLeu Ile Val Phe Val 290 295 5 1053 DNA Homo sapiens CDS (1)..(1050) 5atg gaa caa cgg gga cag aac gcc ccg gcc gct tcg ggg gcc cgg aaa 48 MetGlu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys 1 5 10 15agg cac ggc cca gga ccc agg gag gcg cgg gga gcc agg cct ggg ctc 96 ArgHis Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu 20 25 30 cgggtc ccc aag acc ctt gtg ctc gtt gtc gcc gcg gtc ctg ctg ttg 144 Arg ValPro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45 gtc tcagct gag tct gct ctg atc acc caa caa gac cta gct ccc cag 192 Val Ser AlaGlu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60 cag aga gtggcc cca caa caa aag agg tcc agc ccc tca gag gga ttg 240 Gln Arg Val AlaPro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu 65 70 75 80 tgt cca cctgga cac cat atc tca gaa gac ggt aga gat tgc atc tcc 288 Cys Pro Pro GlyHis His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95 tgc aaa tat ggacag gac tat agc act cac tgg aat gac ctc ctt ttc 336 Cys Lys Tyr Gly GlnAsp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105 110 tgc ttg cgc tgcacc agg tgt gat tca ggt gaa gtg gag cta agt ccc 384 Cys Leu Arg Cys ThrArg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125 tgc acc acg accaga aac aca gtg tgt cag tgc gaa gaa ggc acc ttc 432 Cys Thr Thr Thr ArgAsn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 cgg gaa gaa gattct cct gag atg tgc cgg aag tgc cgc aca ggg tgt 480 Arg Glu Glu Asp SerPro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 160 ccc aga gggatg gtc aag gtc ggt gat tgt aca ccc tgg agt gac atc 528 Pro Arg Gly MetVal Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175 gaa tgt gtccac aaa gaa tca ggt aca aag cac agt ggg gaa gcc cca 576 Glu Cys Val HisLys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro 180 185 190 gct gtg gaggag acg gtg acc tcc agc cca ggg act cct gcc tct ccc 624 Ala Val Glu GluThr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205 tgt tct ctctca ggc atc atc ata gga gtc aca gtt gca gcc gta gtc 672 Cys Ser Leu SerGly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val 210 215 220 ttg att gtggct gtg ttt gtt tgc aag tct tta ctg tgg aag aaa gtc 720 Leu Ile Val AlaVal Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 ctt ccttac ctg aaa ggc atc tgc tca ggt ggt ggt ggg gac cct gag 768 Leu Pro TyrLeu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255 cgt gtggac aga agc tca caa cga cct ggg gct gag gac aat gtc ctc 816 Arg Val AspArg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu 260 265 270 aat gagatc gtg agt atc ttg cag ccc acc cag gtc cct gag cag gaa 864 Asn Glu IleVal Ser Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280 285 atg gaagtc cag gag cca gca gag cca aca ggt gtc aac aaa acc ggg 912 Met Glu ValGln Glu Pro Ala Glu Pro Thr Gly Val Asn Lys Thr Gly 290 295 300 cga gatgcc tct gtc cac acc ctg ctg gat gcc ttg gag acg ctg gga 960 Arg Asp AlaSer Val His Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly 305 310 315 320 gagaga ctt gcc aag cag aag att gag gac cac ttg ttg agc tct gga 1008 Glu ArgLeu Ala Lys Gln Lys Ile Glu Asp His Leu Leu Ser Ser Gly 325 330 335 aagttc atg tat cta gaa ggt aat gca gac tct gcc atg tcc taa 1053 Lys Phe MetTyr Leu Glu Gly Asn Ala Asp Ser Ala Met Ser 340 345 350 6 350 PRT Homosapiens 6 Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala ArgLys 1 5 10 15 Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg ProGly Leu 20 25 30 Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val LeuLeu Leu 35 40 45 Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu AlaPro Gln 50 55 60 Gln Arg Val Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser GluGly Leu 65 70 75 80 Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg AspCys Ile Ser 85 90 95 Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn AspLeu Leu Phe 100 105 110 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu ValGlu Leu Ser Pro 115 120 125 Cys Thr Thr Thr Arg Asn Thr Val Cys Gln CysGlu Glu Gly Thr Phe 130 135 140 Arg Glu Glu Asp Ser Pro Glu Met Cys ArgLys Cys Arg Thr Gly Cys 145 150 155 160 Pro Arg Gly Met Val Lys Val GlyAsp Cys Thr Pro Trp Ser Asp Ile 165 170 175 Glu Cys Val His Lys Glu SerGly Thr Lys His Ser Gly Glu Ala Pro 180 185 190 Ala Val Glu Glu Thr ValThr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205 Cys Ser Leu Ser GlyIle Ile Ile Gly Val Thr Val Ala Ala Val Val 210 215 220 Leu Ile Val AlaVal Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 Leu ProTyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255 ArgVal Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu 260 265 270Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280285 Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Lys Thr Gly 290295 300 Arg Asp Ala Ser Val His Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly305 310 315 320 Glu Arg Leu Ala Lys Gln Lys Ile Glu Asp His Leu Leu SerSer Gly 325 330 335 Lys Phe Met Tyr Leu Glu Gly Asn Ala Asp Ser Ala MetSer 340 345 350

1. An isolated nucleic acid encoding a mammalian TRAIL receptor selectedfrom the group consisting of DR5 and TRAIL-R3, splice variant cDNAsequences thereof, or an active fragment thereof.
 2. The nucleic acidaccording to claim 1, wherein said mammalian TRAIL receptor is isolatedfrom a mammal selected from the group consisting of human, rat, mouse,porcine, ovine, canine and bovine.
 3. The nucleic acid according toclaim 1 wherein the encoded splice variant is DR5s, or an activefragment thereof.
 4. The nucleic acid of claim 3 comprising SEQ ID NO:5or an active fragment thereof.
 5. A nucleic acid encoding a mammalianTRAIL receptor, wherein said nucleic acid is selected from the groupconsisting of: (a) DNA encoding the amino acid sequence set forth in SEQID NO:2, SEQ ID NO:4, or SEQ ID NO:6; (b) DNA that hybridizes to the DNAof (a) under moderately stringent conditions, wherein said DNA encodesbiologically active DR5 or TRAIL-R3; (c) DNA degenerate with respect toeither (a) or (b) above, wherein said DNA encodes biologically activeDR5,or TRAIL-R3; and (d) splice variant cDNA sequences of any of(a)-(d).
 6. The nucleic acid according to claim 5 wherein the splicevariant cDNA comprises SEQ ID NO:5.
 7. The nucleic acid according toclaim 5, wherein said nucleic acid hybridizes under high stringencyconditions to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
 8. The nucleicacid according to claim 5, wherein the nucleotide sequence of saidnucleic acid is substantially the same as nucleotides set forth in SEQID NO: 1, SEQ ID NO:3, or SEQ ID NO:5.
 9. A nucleic acid according toclaim 5, wherein the nucleotide sequence of said nucleic acid is SEQ IDNO: 1, SEQ ID NO:3, or SEQ ID NO:5.
 10. A nucleic acid according toclaim 5, wherein said nucleic acid is cDNA.
 11. A vector containing thenucleic acid of claim
 5. 12. An expression vector comprising the nucleicacid of claim 5, wherein the nucleic acid encoding the TRAIL receptor isoperatively linked to a promoter.
 13. Recombinant cells containing thenucleic acid of claim
 5. 14. An antisense oligonucleotide capable ofspecifically binding to mRNA encoded by said nucleic acid according toclaim
 5. 15. An isolated mammalian protein selected from the groupconsisting of DR5, a DR5 splice variant, TRAIL-R3, and a TRAIL-R3 splicevariant, wherein said protein is characterized by being able to bindTRAIL ligand.
 16. The protein according to claim 15, wherein the aminoacid sequence of said protein comprises substantially the same sequenceas the protein sequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:6.
 17. The protein according to claim 16, comprising the sequence setforth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
 18. The proteinaccording to claim 15, wherein said protein is encoded by a nucleotidesequence that is substantially the same as SEQ ID NO: 1, SEQ ID NO:3, orSEQ ID NO:5.
 19. The protein according to claim 18, wherein said proteinis encoded by a nucleotide sequence set forth as SEQ ID NO: 1, SEQ IDNO:3, or SEQ ID NO:5.
 20. A method for expression of a protein selectedfrom the group consisting of DR5, a DR5 splice variant, TRAIL-R3, and aTRAIL-R3 splice variant, said method comprising culturing cells of claim13 under conditions suitable for expression of said protein.
 21. Anisolated anti-DR5 or anti-TRAIL-R3 antibody having specific reactivitywith a protein according to claim
 15. 22. The antibody according toclaim 21, wherein said antibody is a monoclonal antibody.
 23. Theantibody according to claim 21, wherein said antibody is a polyclonalantibody.
 24. A composition comprising an amount of the antisenseoligonucleotide according to claim 14 effective to inhibit expression ofa human DR5 or TRAIL-R3 protein and an acceptable hydrophobic carriercapable of passing through a cell membrane.
 25. A transgenic non-humanmammal expressing exogenous nucleic acid encoding a DR5 or TRAIL-R3protein according to claim
 15. 26. A transgenic non-human mammalaccording to claim 25, wherein the transgenic non-human mammal is amouse.
 27. A method for detecting the presence of a mammalian DR5 orTRAIL-R3 protein in a sample, said method comprising contacting a testsample with an antibody according to claim 21, detecting the presence ofan antibody-DR5 complex or antibody-TRAIL-R3 complex, and therefromdetecting the presence of a mammalian DR5 or TRAIL-R3 protein in saidtest sample.
 28. A bioassay for evaluating whether test compounds arecapable of acting as agonists or antagonists for DR5 or TRAIL-R3proteins according to claim 15, said bioassay comprising: (a) culturingcells containing: DNA which expresses DR5 or TRAIL-R3 proteins orfunctional modified forms thereof, wherein said culturing is carried outin the presence of at least one compound whose ability to modulateapoptotic activity of DR5 or TRAIL-R3 protein is sought to bedetermined, and thereafter (b) monitoring said cells for either anincrease or decrease in the level of apoptosis.
 29. A bioassay forevaluating whether test compounds are capable of acting as antagonistsfor DR5 or TRAIL-R3 proteins according to claim 18, or functionalmodified forms of said DR5 or TRAIL-R3 proteins, said bioassaycomprising: (a) culturing cells containing: DNA which expresses DR5 orTRAIL-R3 proteins, or functional modified forms thereof, wherein saidculturing is carried out in the presence of: increasing concentrationsof at least one compound whose ability to inhibit apoptotic activity ofDR5 or TRAIL-R3 proteins is sought to be determined, and a fixedconcentration of TRAIL; and thereafter (b) monitoring in said cells thelevel of apoptosis as a function of the concentration of said compound,thereby indicating the ability of said compound to inhibit DR5 orTRAIL-R3 apoptotic activity.
 30. A method for modulating the apoptoticactivity mediated by DR5 or TRAIL-R3 protein, said method comprising:contacting said DR5 or TRAIL-R3 protein with an effective, modulatingamount of said agonist or antagonist identified by claim 28.